Sugar transporters

ABSTRACT

A novel class of transporter protein, referred to as SWEET, GLUE or Glü, is disclosed. These transporters provide a novel system for the transportation of sugars across membranes within a cell and between the inside and outside of a cell. Such transporters are useful for understanding and altering the sugar concentration within certain organs of an organism, and within certain organelles within the cell. These transporters are also useful in protecting plants from a pathogen attack.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/175,267 (filed May 4, 2009), which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled “056100-5077-SeqListing.txt,”created on or about Oct. 20, 2010 with a file size of about 2,455 kbcontains the sequence listing for this application and is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel sugar transporters across themembrane of a cell.

BACKGROUND OF THE INVENTION

The need and use of carbohydrates in many biochemical pathways has beenextensively studied and reported over centuries. Mono-, di-, andpolysaccharides, or sugars, are a prime dietary source of carbohydratefor many organisms.

Glucose is one of the more readily available sugars and its structurelends itself to be readily acted on by the biochemical systems of manyorganisms. Comprised of six carbon atoms, glucose falls within thecategory of aldehexoses. Aldehexose has four chiral centers which leadto 16 stereoisomers. Two stereoisomers of aldehexoses are regarded asglucoses, the major one being D-glucose. All major dietary carbohydratescontain glucose, either as their only building block, as in starch andglycogen, or together with another monosaccharide, as in sucrose andlactose. The metabolism of this carbohydrate translates into energy,such as adenosine triphosphate (ATP). Other metabolic routes for glucoselead to energy storage. The glucose molecule can exist as an open-chain(acyclic) form or in a ring (cyclic) form.

Glucose may be used as a precursor for the synthesis of severalimportant substances, such as starch, cellulose, and glycogen. Lactose,a sugar in milk, is a glucose-galactose disaccharide. Sucrose, anotherdisaccharide, joins glucose to fructose. While glucose is the majortransport form of sugars in metazoa, sucrose and its derivatives servesas the major transport form in plants.

Glucose is one of the downstream products of photosynthesis in plantsand some prokaryotes. In eukaryotes, such as animals and fungi, glucosemay be produced as the result of the breakdown of glycogen, through aprocess referred to as glycogenolysis. In plants, the resultingbreakdown substrate is starch. Glucose may also be derived from theaction of invertase on the major transport sugar sucrose in plants (inthe cell wall, the cytosol or vacuole, each by a specific isoform).

In animals, glucose may be synthesized in the liver and kidneys fromnon-carbohydrate intermediates, such as pyruvate and glycerol through aprocess referred to as gluconeogenesis. Glucose may also be synthesized,such as through enzymatic hydrolysis of starch. Commercially, crops suchas maize, rice, wheat, potato, cassava, sago, and arrowroot may be usedas a source of starch.

Glucose may be used in either aerobic or anaerobic respiration.Carbohydrates are a significant source of energy for organisms. Aerobicrespiration can provide roughly 3.75 kcal of energy per gram. Breakdownof carbohydrates, such as starch, results in monosaccharides anddisaccharides. Through the process of glycolysis and the reactions ofthe citric acid cycle (or Krebs cycle), glucose is oxidized and brokendown to eventually forms carbon dioxide and water, yielding energysources, predominantly ATP. The insulin reaction, as well as othermechanisms, may regulate the concentration of glucose in the blood.

The need for energy in neurological centers, such as the brain, directlycorrelates glucose to psychological processes. Glucose is a primarysource of energy for the brain, and hence its availability influencespsychological processes. When glucose is low, psychological processesrequiring mental effort may be impaired. Both aerobic and anaerobicrespiration start with the early steps of the glycolysis metabolicpathway, the first step being the phosphorylation of glucose byhexokinase to prepare it for later breakdown to provide energy. Theimmediate phosphorylation of glucose by a hexokinase may then preventdiffusion out of the cell. The act of phosphorylation adds a chargedphosphate group, thereby preventing the glucose-6-phosphate from easilycrossing the cell membrane. Glucose is also important for the productionof proteins and in the process of lipid metabolism. Glucose may alsoserve as a precursor molecule for ascorbic acid, or vitamin C.

Accordingly, the uptake, absorption, processing, metabolism, exchangeand transport of sugars, such as glucose and sucrose, within a cell andbetween cells of a tissue in an organism is of utmost importance for theability of a cell or the organism comprising the cell to thrive.Dysfunction of glucose or sucrose transport across cell membranes andbetween the organelles of a cell can be catastrophic. There is a need todevelop methods to regulate the transport of glucose efficiently.

SUMMARY OF THE INVENTION

The present invention provides a novel class of protein transporters fortransporting sugars in a cell. The transporters, referred to as GLUEs(also known as Glüs or SWEETs), may be in a plant and may be encoded bya nucleic acid encoding a sugar transporter (e.g. pentose, glucose,mannose, in sum monosaccharides), or sucrose and maltose (in sum di- andoligosaccharides) having at least 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, or 99% sequence identity with the following accession nos:AT4G15920 (SEQ ID NO: 1), AT3G16690 (SEQ ID NO: 2), AT5G13170 (SEQ IDNO: 3), AtSAG29 (SEQ ID NO: 4), AT4G25010 (SEQ ID NO: 5), AT5G50800 (SEQID NO: 6), AT5G23660 (SEQ ID NO: 7), AT3G48740 (SEQ ID NO: 8), AT5G50790(SEQ ID NO: 9), AT2G39060 (SEQ ID NO: 10), AT5G40260 (SEQ ID NO: 11),AtRPG1 (SEQ ID NO: 12), AT4G10850 (SEQ ID NO: 13), AT1G66770 (SEQ ID NO:14), AT1G21460 (SEQ ID NO: 15), AT5G62850 (SEQ ID NO: 16), AtVEX1 (SEQID NO: 17), AT3G28007 (SEQ ID NO: 18), AT3G14770 (SEQ ID NO: 19),AT1G21460 (SEQ ID NO: 15), AT5G53190 (SEQ ID NO: 20), NEC1 (SEQ ID NO:21 and SEQ ID NO: 21), AC202585 (SEQ ID NO: 22), AC147714 (SEQ ID NO:23), MtC60432 GC (SEQ ID NO: 25 and SEQ ID NO: 26), MtC11004 GC (SEQ IDNO: 27 and SEQ ID NO: 28), CT963079 (SEQ ID NO: 29), MtD03138 GC (SEQ IDNO: 30), TC 125536 (SEQ ID NO: 31 and SEQ ID NO: 32), AC146866 (SEQ IDNO: 33), AC189276 (SEQ ID NO: 34), CAA69976 (SEQ ID NO: 35), AC2456 (SEQID NO: 36), TC115479 (SEQ ID NO: 37), AC146747 (SEQ ID NO: 38), MtC10424GC (SEQ ID NO: 39), CT954252 (SEQ ID NO: 40 and SEQ ID NO: 41), CU302340(SEQ ID NO: 42 AC202585 (SEQ ID NO: 43), AC147714 (SEQ ID NO: 44),MtC60432 GC (SEQ ID NO: 45 and SEQ ID NO: 46), MtC11004 GC (SEQ ID NO:47 and SEQ ID NO: 48), CT963079 (SEQ ID NO: 49), Os08g42350 (Os8N3) (SEQID NO: 50 and SEQ ID NO: 51), Os08g0535200 (SEQ ID NO: 52 and SEQ ID NO:53), Os12g29220 (SEQ ID NO: 54 and SEQ ID NO: 55), Os03g0347500 (SEQ IDNO: 56 and SEQ ID NO: 57), Os05g51090 (SEQ ID NO: 58 and SEQ ID NO: 59),Os05g0588500 (SEQ ID NO: 58 and SEQ ID NO: 59), Os12g07860 (SEQ ID NO:60 and SEQ ID NO: 61), Os09g08440 (SEQ ID NO: 62 and SEQ ID NO: 63),Os09g08490 (SEQ ID NO: 64 and SEQ ID NO: 65), Os09g08270 (SEQ ID NO: 66and SEQ ID NO: 67), Os09g08030 (SEQ ID NO: 68 and SEQ ID NO: 69),Os09g0254600 (SEQ ID NO: 68 and SEQ ID NO: 69), Os01g42090.1 (SEQ ID NO:70 and SEQ ID NO: 71), Os01g0605700 (SEQ ID NO: 70 and SEQ ID NO: 71),Os01g42110.1 (SEQ ID NO: 72 and SEQ ID NO: 73), Os01g060600 (SEQ ID NO:72 and SEQ ID NO: 73), Os02g19820 (SEQ ID NO: 74 and SEQ ID NO: 75),Os02g0301100 (SEQ ID NO: 74 and SEQ ID NO: 75), Os05g35140 (SEQ ID NO:76 and SEQ ID NO: 77), Os05g0426000 (SEQ ID NO: 76 and SEQ ID NO: 77),Os01g65880 (SEQ ID NO: 78 and SEQ ID NO: 79), Os01g0881300 (SEQ ID NO:78 and SEQ ID NO: 79), Os01g50460 (SEQ ID NO: 80 and SEQ ID NO: 81),Os01g0700100 (SEQ ID NO: 80 and SEQ ID NO: 81), Os01g36070.1 (SEQ ID NO:82 and SEQ ID NO: 83), Os01g0541800 (SEQ ID NO: 82 and SEQ ID NO: 83),Os01g12130.1 (SEQ ID NO: 84 and SEQ ID NO: 85), Os05g12320 (SEQ ID NO:86 and SEQ ID NO: 87), Os05g0214300 (SEQ ID NO: 88 and SEQ ID NO: 89),and Os01g21230 (SEQ ID NO: 88 and SEQ ID NO: 89) (all of which areherein incorporated by reference in their entirety). The nucleic acidmay be in a vector and/or in a cell, such as a plant cell or an animalcell. The present invention also provides transgenic plants comprisingthe GLUEs. The nucleic acid may be encoded by a nucleic acid encoding aglucose or sucrose transporter having at least 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, or 99% sequence identity with the following animalaccession nos: (e.g. from the worm C. elegans) R10D12.9, K11D12.5, andK06A4.4, K02D7.5, C54F6.4, C06G8.1, Y39A1A.8, ci-rga, RAG1AP1 (e.g.,from Drosphila, human, mouse, Rattus norvegicus, and Xenopus) (all ofwhich are herein incorporated by reference in their entirety).

The present invention further provides mutated GLUE proteins. A GLUE maybe mutated so that the passage of sugar through the GLUE is improved ascompared to a wild type. The mutations may improve the functioning of aGLUE so that more sugar can be transported, either through increasedrate of passage or through an increased capacity for transport. Themutation may prevent or impede the passage of sugar through the GLUE ascompared to the wild type. The mutation may be a deletion orsubstitution of an amino acid or amino acids in the wild type sequence.The mutation may be a truncation of the GLUE.

The present invention provides fusion proteins comprising a GLUEprotein. The GLUE may be fused to a tag, such as an epitope. The GLUEmay be part of a chimeric membrane protein, such as other seventransmembrane protein with known downstream cascades. The chimericprotein may comprise replacing the third intracellular loop and/or thecytoplasmic tail of the GLUE with the corresponding domains from anotherseven transmembrane protein. The GLUE may be coupled to a targetingsequence to direct expression and location of the GLUE to a particularorganelle or region within a cell. The GLUE may be a mutated GLUEprotein.

The present invention provides methods of generating a plant thatproduces an increased level of carbon as compared to a control plantcomprising introducing a nucleic acid encoding a mono-, di- oroligosaccharide transporter into a plant cell and growing the plant cellinto a plant that expresses the nucleic acid, wherein the nucleic acidencodes a GLUE.

The present invention provides methods of increasing transport of sugarinto the root of a plant comprising introducing into a cell of the planta nucleic acid encoding a GLUE. The introduction of a GLUE into a plantmay provide methods for modulating sugar secretion into the rhizosphereof a plant and methods for modulating transport of sugar into thephyllosphere of a plant or the delivery of sugars to developing seeds,flowers etc.

The present invention provides methods for altering the levels of sugarin a plant comprising introducing a nucleic acid encoding a GLUE into acell. The methods may increase sugar levels within a cell. The methodsmay decrease sugar levels within a cell. The methods may direct sugarconcentration to accumulate in certain regions, organs or organelles ina plant or animal. The methods may cause a decline in sugar levels incertain regions, organs, or organelles in a plant or animal. The methodsmay increase sugar import. The methods may decrease sugar import. Themethods may increase sugar export. The methods may decrease sugarexport. The GLUEs may be expressed in a cell with a cofactor, such asanother intracellular protein or another transporter, such as acotransporter.

The present invention provides methods of attracting beneficialmicroorganisms to a plant comprising altering the sugar concentrationthrough the introduction of a GLUE. The present invention furtherprovides methods of protecting a plant from a pathogen through theintroduction of a GLUE. Pathogens attacking a plant may utilize theplant's cell machinery to alter sugar exportation in the plant. Byintroducing into the plant an exogenous GLUE, which may further be underthe control of a different promoter, the pathogen's ability to altersugar exportation may be limited or altered.

The present invention also provides methods for determining how a GLUEis acting within a cell or an organism. An exogenous GLUE may beco-expressed in a cell with a sugar detecting molecule, such as aprotein comprising a sugar (e.g. glucose or sucrose) binding domainsandwiched between a fluorescent donor domain and a fluorescent acceptordomain. The concentration of sugar may be determined and monitored overtime through the use of fluorescent resonance energy transfer.

The present invention provides methods for affecting and/or altering theexpression of glucose transporter facilitator (“GLUT”) proteins in acell.

The present invention provides for methods of altering the sugar levelwithin a fluid secreted by a cell, such as nectar or milk. The presentinvention provides methods for altering the development of an organismby introducing a GLUE into a cell in the organism. The GLUE may bemutated. The present invention provides methods for altering thedevelopment of an organism by mutating a GLUE in a cell in the organism.The increased or decreased functioning of a GLUE within an organism mayalter sugar concentrations and/or sugar distribution through the celland throughout the organism and thereby affect development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phylogenetic tree of the GLUE superfamily cDNA in variousplant species. FIG. 1A shows a phylogenetic tree of the GLUEsuperfamily. Distances were calculated from a multiple sequencealignment (ClustalW) using the neighbor-joining method and the treedisplays the bootstrap values (percentage of 1000). The SWEET family canbe divided into 4 clades. All sequences were obtained from NCBI or theAramemnon database. The trees for rice, arabidopsis, medicago, andpetunia are illustrated. The scale shown represents a change of 50 basesper length illustrated.

FIG. 2 shows an overview of various means by which sugars aretransported and move within a cell and with the organs and organelles ofa plant.

FIGS. 3A and 3B show the response with a glucose FRET sensor expressedin a cell. FIG. 3A shows the response of the sensor to variousconcentrations of glucose without the co-expression of a GLUE. FIG. 3Bshows that introducing GLUE1 into a cell with the glucose FRET sensorresults in significant changes to the sensor when the concentration ofsugar is altered.

FIGS. 4A-4D show the response of the glucose FRET sensor to alteredconcentrations of glucose with varying GLUE proteins expressed. FIG. 4Ashows the response with GLUE1. FIG. 4B shows the response with GLUE12.FIG. 4C shows the response with GLUE8. FIG. 4D shows the response withGLUE13.

FIG. 5 shows that the glucose uptake-deficient strain EBY4000 withvector only (EBY-pDRF1) can not uptake D-glucose. Radiolabelled14C-glucose uptake was measured over time. In contrast, GLUE1 enablesyeast strain EBY4000 (EBY-GLUE1) to take up glucose. The glucose uptakecompetent wild type strain CENPK (CEN) serves as a positive control andis able to take up glucose.

FIG. 6 shows that 4 of 15 genes for GLUEs tested can rescue yeastmutant. Yeast was grown on 2% glucose with different pH.

FIG. 7 shows the induction of RAG1AP1 (HsSWEET1) during lactation.Microarray data suggest that the putative sugar transporter RAG1AP1 isupregulated during lactation.

FIG. 8 shows lactose synthesis and secretion from alveolar cells.Glucose is imported through the basal membrane by GLUTs/SGLTs and thenimported either into the ER (circle 1) or Golgi (circle 2) by unknowntransporters. Lactose synthesis occurs in the Golgi and lactose isassumed to be exocytosed on the apical side that faced the milk duct. Anunknown transporter either exports glucose through the apical membraneor is involved in retrieval of glucose from the milk (circle 3).

FIG. 9 shows the identification and characterization of SWEET (GLUE)transporters. FIG. 9A shows the identification of glucose transportactivity for SWEET1 by coexpression with the cytosolic FRET glucosesensor FLIPglu600μΔ13V in HEK293T cells. Individual cells were analyzedby quantitative ratio imaging of CFP and Venus emission (acquisitionintervals 5 sec; Fc/D corresponds to the normalized emission intensityratio). HEK293T/FLIPglu600μΔ13V cells were perfused with medium,followed by square pulses of increasing glucose concentrations. Cellsexpressing only the sensor did not accumulate significant amounts ofglucose in the cytosol as indicated by a lack of a FRET ratio change(orange line). Cells coexpressing the sensor and SWEET1 accumulatedglucose as evidenced by the negative FRET ratio change with an amplitudethat correlates with the increasing external glucose supply (blue line).Data points are mean±SD (n>10). FIG. 9B shows FRET imaging of ‘efflux’of glucose from the cytosol into the ER (cf. FIG. 9C). The sensorFLIPglu600μΔ13V^(ER) was targeted to the lumen of the ER (analysisperformed as under FIG. 9A, acquisition intervals 10 sec). Cellsexpressing only the sensor did not accumulate significant amounts ofglucose in the ER. Cells coexpressing the sensor and SWEET1 accumulatedglucose in the ER as evidenced by the negative ratio change induced byperfusion with glucose. Data points are mean±SD (n>10). FIG. 9C shows acartoon for SWEET1 influx across the PM and efflux from cytosol to ER.The cytosolic sensor FLIPglu600μΔ13V identifies transport of glucoseinitiated at the extracellular face (indicated by extracellularN-terminus). FLIPglu600μΔ13V^(ER) measures transport initiated at theintracellular side (cytosolic C-terminus). FIG. 9D shows acomplementation of yeast strain EBY4000 lacking all 18 hexosetransporter genes by SWEET1, SWEET8, or yeast HXT5; control: emptyvector. FIG. 9E shows accumulation of glucose in EBY4000 coexpressingSWEET1 and FLII¹²Pglu700ξδ6 before and after addition of 0, 20 and 100mM glucose. Two cycles were run before addition of glucose. Data aremean±SD, n=3. FIG. 9F shows kinetics of ¹⁴C-glucose accumulation bySWEET1 in EBY4000. Data are mean±SD, n=3. FIG. 9G shows confocal imagingof SWEET1-YFP in leaves of stably transformed Arabidopsis leaves. FIG.9H shows structural model of SWEETs based on hydrophobicity plots. Eachprotein contains seven TMHs with a predicted extracellular N-terminusand a predicted parallel orientation of two ‘subunits’ derived from aduplication of three TMHs (TMH1-3 and 5-7, highlighted by red and bluetriangles), separated by TMH4 as linker. FIG. 9I shows uptake of[¹⁴C]-glucose into Xenopus oocytes mediated by SGLT1, but not byOsSWEET11. Coexpression of OsSWEET11 with SGLT1 reduces glucoseaccumulation in oocytes. Data are mean±SE, n=7. Inset indicatesconcentrative uptake of glucose by SGLT1 and glucose efflux (‘leak’)caused by OsSWEET11. FIG. 9J shows direct efflux measurements fromoocytes expressing SWEET1 or OsSWEET11. 50 nl of 10 mM radiolabeledglucose (0.18 μCi/μl) were injected and radiotracer efflux was measuredover time. Data are mean±SE (n>10 cells).

FIG. 10 shows biotrophic bacteria or fungi induce mRNA levels ofdifferent SWEET genes. FIG. 10A shows induction of SWEET mRNAs by eitherthe bacterium Pseudomonas syringae pv. tomato DC3000 (2×10⁸ cfu/ml, 8hrs post inoculation, measured by qPCR, normalized by MgCl₂ buffertreatment), the powdery mildew fungus, G. cichoracearum, (˜25-35conidiospores mm⁻², 48 hrs post inoculation, measured by qPCR;normalized to 0 hr values), or by the fungus Botytis cinerea) inArabidopsis leaves. FIG. 10B shows induction of SWEET4, 5 and 15 by P.s.DC3000 depends on a functional type III secretion system (T3S). Sampleswere collected at 6, 12 and 24 hr after infiltration with 2×10⁸ cfu/mlof DC3000 or DC3000 ΔhrcU, a T3S mutant. FIG. 10C shows infection by G.cichoracearum leads to induction of SWEET11 and SWEET12 butdown-regulation of SWEET15. Samples were taken after 0, 8, 12, 24 and 72hr post-inoculation.

FIG. 11 shows a schematic model for the role of SWEETs in nutrition ofpathogens. FIG. 11A shows the pathogenic bacterium Xanthomonas oryzaepv. oryzae strain PXO99^(A)(X.o. PXO99^(A)) injects the TAL effectorPthXo1 via type III secretion system into rice cells. Thistranscriptional activator directly or indirectly triggers induction ofthe rice OsSWEET11/Os8N3 glucose efflux transporter gene leading tosecretion of glucose. Bacteria take up glucose via endogenous uptakesystems and can multiply. FIG. 11B shows that if PthXo1 is mutated (ME),induction of OsSWEET11/Os8N3 is reduced or abolished, leading tostarvation of the bacteria (indicated as reduced size of bacterial cell,meant low cell number). FIG. 11C shows mutation of OsSWEET11/Os8N3 alsoleads to starvation of bacteria. FIG. 11D shows a pathogen expressing analternative effector AvrXa7 can multiply if it induces another member ofthe SWEET family (or by inducing access to another carbon source).

FIG. 12 shows evidence for SWEET-mediated glucose transport in HEK293Tcells. FIG. 12A shows inhibition of GLUT1 activity by 20 μM cytochalasinB analyzed using the FLIPglu600μΔ13V sensor co-transfected with GLUT1.FIG. 12B shows insensitivity of SWEET1 activity to 20 μM cytochalasin Banalyzed using the FLIPglu600μΔ13V sensor co-transfected with SWEET1.FIG. 12C shows expression level of SLC2 (GLUT) and SLC5 (SGLT) glucosetransporter genes in HepG2 cells, HEK293T cells, and HEK293T cellscoexpressing FLIPglu600μΔ13V and/not SWEET1.

FIG. 13 show biochemical properties of SWEET1. FIG. 13A shows pH optimumfor SWEET1. Radiotracer uptake was measured at different pH. The pHoptimum for uptake is about pH 8.5. Data are mean±S.D. FIG. 13B showsinhibition of glucose uptake (5 mM D-glucose; 0.1 μCi [¹⁴C]-D-glucose)mediated by SWEET1 in the yeast strain EBY4000 by different sugars.Competitors were added at 10-fold excess (final concentration 50 mM).Relative activity was normalized to D-glucose uptake rate [100%]. Dataare mean±S.D.

FIG. 14 shows tissue specific expression pattern of SWEET1 and SWEET8genes in Arabidopsis. The analysis is based on microarray studies fromthe Arabadopsis eFP Browser, which is available on the internet at“bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi”.

FIG. 15 shows a functional analysis of SWEET8 in heterologous systems.FIG. 15A shows cells coexpressing the cytosolic FRET glucose sensorFLIPglu600μΔ13V and SWEET8 accumulated glucose in the cytosol asevidenced by a negative cytosolic FRET ratio change in HEK293T cells(cf. FIG. 9A). FIG. 15B shows cells ‘efflux’ glucose from the cytosolinto the ER when coexpressing the ER FRET glucose sensorFLIPglu-600μΔ13V^(ER) and SWEET8 in HEK293T cells (cf. FIG. 9B). Datapoints are mean±SD (n>10). FIG. 15C shows relative uptake rate ofSWEET1, SWEET8 and vector control in the yeast glucosetransport-deficient mutant EBY4000 (2 min; 10 mM D-glucose; 0.1 μCi[¹⁴C]-D-glucose). Values are normalized to SWEET1 (100%). Data aremean±S.D. FIG. 15D shows confocal imaging of SWEET8 localization inseedlings using stably transformed Arabidopsis (T1 generation).

FIG. 16 shows SWEET-mediated glucose uptake in Xenopus oocytes. Relativeglucose uptake rate in oocytes injected with water as control, SWEET1,OsSWEET11, SGLT1 or SGLT1 together with SWEET1 (time=1 h; 1 mMD-glucose; 4 μCi/ml [¹⁴C]-D-glucose). Data are mean±SD, n>9.

FIG. 17 shows real time accumulation of glucose in HEK293T cells. GLUE1was coexpressed with cytosolic or ER FRET sensor FLIPglu600μΔ6 inHEK293T cells. The normalized emission ratio of CFP and YFP is shown onthe Y-axis. Negative FRET ratio changes indicate that GLUE1 function asglucose transporter and effluxer as well.

FIG. 18 shows GLUE8, 12, and 13 were coexpressed with cytosolic FRETsensor FLIPglu600μΔ6 in HEK293T cells. The normalized emission ratio ofCFP and YFP is shown on the Y-axis. Negative FRET ratio changes incytosol indicate that GLUE8, 12, and 13 function as glucose transporterswith different activities.

FIG. 19 shows functional expression of GLUEs in Xenopus ooxytes. FIG.19A shows uptake of [14C]-glucose into Xenopus oocytes mediated bySGLT1, GLUE1, but not by OsGLUE11. Coexpression of OsGLUE11 with SGLT1reduces glucose accumulation. FIG. 19B shows uptake of ¹⁴C-glucose intoXenopus oocytes mediated by CeSWEET1, 3, 4, 5, 7 and RAG1AP1 splicevariants 1, and 2 and a mutated version (Y216A, L218A, L219A;RAG1AP1-3aa). SGLT1 served as control. FIG. 19C shows the effect ofcoexpression of CeSWEETs and RAG1AP1 variants on glucose accumulation bySGLT1 in Xenopus oocytes. All experiments were repeated independently atleast 6 times. Error bars are means±SD.

FIG. 20 shows complementation of yeast strain YSL2-1 lacking all 18hexose transporter genes with 17 Arabidopsis GLUE genes. Cellsexpressing yeast homolog, HXT5, or mammalian homolog, GLUT1, were usedas controls. Os8N3 and RAG1AP1 were homologs from rice and mammalian.The yeast cells were grown in SD-Ura liquid medium with 2% maltose toearly log phase and 5 m of serious dilutions were spotted on the mediacontaining YPM or SD-URA containing 2% of maltose, fructose, or mannose.GLUE4 and GLUE7 transport fructose and mannose. GLUE5 and 8 transportmannose.

FIG. 21 shows complementation of yeast strain YSL2-1 lacking all 18hexose transporter genes with 17 Arabidopsis GLUE genes. Cellsexpressing mammalian homolog, GLUT1, were used as controls. Os8N3 andRAG1AP1 were homologs from rice and mammalian. The yeast cells weregrown in SD-Ura liquid medium with 2% maltose to early log phase and 5 mof serious dilutions were spotted on the media containing SD-URAcontaining various concentrations of galactose. Except for GLUE1, 4, 5,and 7, all others are sensitive to 5% galactose, indicating thecapability to mediate galactose transport.

FIG. 22 shows complementation of yeast strain YSL2-1 lacking all 18hexose transporter genes with 17 Arabidopsis GLUE genes. Cellsexpressing yeast homolog, HXT5, or mammalian homolog, GLUT1, were usedas controls. Os8N3 and RAG1AP1 were homologs from rice and mammalian.The yeast cells were grown in SD-Ura liquid medium with 2% maltose toearly log phase and 5 m of serious dilutions were spotted on the mediacontaining YPM or SD-URA containing 2% glucose. GLUE1, 4, 5 and 7transport glucose (GLUE8 as well, but not shown here).

FIG. 23 shows complementation of yeast strain YSL2-1 lacking all 18hexose transporter genes with 17 Arabidopsis GLUE genes. Cellsexpressing yeast homolog, HXT5, or mammalian homolog, GLUT1, were usedas controls. Os8N3 and RAG1AP1 were homologs from rice and mammalian.The yeast cells were grown in SD-Ura liquid medium with 2% maltose toearly log phase and 5 m of serious dilutions were spotted on the mediacontaining YPM or SD-URA containing various 2-Deoxy-glucose levels.GLUE1, 3, 4, 5, 7, 8, 14, 16 and 17 transport 2-deoxyglucose since theyare more sensitive to the sugar analog.

FIG. 24 shows subcellular localization of Arabidopsis GLUE protein inplanta. GLUE-GFP fusion proteins localize close to or to the plasmamembrane when transiently expressed in tobacco leaves.

FIG. 25 shows GLUE expression in roots. qPCR analysis of GLUE geneexpression in Arabidopsis roots. Transcripts were isolated from10-day-old Arabidopsis seedlings and cDNA was generated as template. Therelative expression levels were calculated using the comparative Ctmethod (1000*1/(2^(CtGIU-CtActin8). Members of the family not shown heredid not show significant expression levels (Guo et al., unpublishedresults). Data from four independent experiments.

FIG. 26 shows histochemical analysis of expression patterns ofArabidopsis GLUE. GUS activity in transgenic Arabidopsis carrying theGLUE2, GLUE16, and GLUE17-GUS fusion proteins was analyzed by stainingwith X-gluc. Images are shown of whole plants from 10-d-old Arabidopsisseedlings.

FIG. 27 shows sugar flux analysis in CIT3 cells with FRET glucosesensor. FRET analysis in CIT3 cell as a human mammary gland cell line,in the absence (A) or the presence (B) of co-expressing RAG1AP1-mCherry,with cytosolic FRET glucose sensor, FLIPglu-30μΔ13V. Cells were perfusedwith different external glucose concentrations (5, 25, and 40 mM). FRETimages were acquired and data were analyzed. Data are mean±SD (n=7-9).

FIG. 28 shows the effect of Cytochalasin B on glucose level in CIT3expressing RAG1AP1mCherry. FRET analysis in CIT3 cell as a human mammarygland cell line, in presence of co-expressing RAG1AP1-mCherry, withcytosolic FRET glucose sensor, FLIPglu-30μΔ13V. Cells were perfused withexternal 40 mM glucose in the presence or absence of 20 μM cytochalasinB. FRET images were acquired and data were analyzed. Data are mean±SD(n=5).

FIG. 29 shows the effect of differentiation on glucose level in CIT3cells expressing RAG1AP1mCherry. FRET analysis in CIT3 cell as a humanmammary gland cell line, in presence of co-expressing RAG1AP1-mCherry,with cytosolic FRET glucose sensor, FLIPglu-30μΔ13V. Cells weredifferentiated by 10 μg/mL insulin, 3 μg/mL prolactin and 3 μg/mLhydrocortisone (secretion medium). Cells were cultured in DMEM/F12containing 10 μg/mL insulin and 5 ng/mL EGF (growth medium). Cells wereperfused with external different glucose concentration (5, 25, and 40mM). FRET images were acquired and data were analyzed. Data are mean±SD(n=11-13).

FIG. 30A shows the localization of RAG1AP1-GFP fusion protein in CIT3cells. The image was taken by confocal microscopy. FIG. 30B shows RT-PCRanalysis of RNA from HepG2 cells and HEK293T cells. RAG1AP1, GLUT1 orβ-actin were reverse transcribed and amplified by PCR.Non-differentiated or differentiated cells were cultured in GM (Growthmedium), DMEM/F12 containing 10 μg/mL insulin and 5 ng/mL EGF or SM(secretion medium), DMEM/F12 containing 10 μg/mL insulin, 3 μg/mLprolactin and 3 μg/mL hydrocortisone.

FIG. 31 shows a western blot of RAG1AP1 and RAG1AP1mCherry. Whole celllysate of yeast, CIT3 with over-expressing RAG1AP1 or RAG1AP1-mCherrywere separated by SDS-PAGE (12.5% gel). Antigen region is EQDRNYWLLQT(SEQ ID NO: 90), corresponding to C terminal amino acids 211-221 ofhuman RAG1AP1 (Abcam).

FIG. 32 shows immunofluorescence localization of RAG1AP1 in MDCK cellsover-expressing RAG1AP1. RAG1AP1 was stained by antibody against theC-terminal peptide of human RAG1AP1 (Abcam) and Alexa 594-labeleddonkey-anti-goat IgG. 2,6-Sialyltransferase-GFP (golgi marker) wasmerged to RAG1AP1.

FIG. 33A shows localization of RAG1AP1-GFP fusion protein in MDCK cells.The image was taken by confocal microscopy. FIG. 33B shows sugar fluxanalysis in HEK293T cells with FRET glucose sensor. FRET analysis inHEK293T cell expressing RAG1AP1 and co-expressing RAG1AP1-mCherry, withcytosolic FRET glucose sensor, FLIPglu-600μΔ13V. Cells were perfusedwith different external glucose concentrations (2.5, 5, 25, and 40 mM).FRET images were acquired and data were analyzed. Data are mean±SD (n=).

FIG. 34 shows immunofluorescence localization of RAG1AP1 in human liversections. RAG1AP1 was stained by antibody against the C-terminal peptideof human RAG1AP1 (Abcam) and Alexa fluor 594-labeled donkey-anti-goatIgG. Golgin-97 was used as golgi-marker, which was stained by monoclonalantibody against golgin-97 (Invitrogen) and Alexa fluor 488-labeleddonkey-anti-mouse IgG.

FIG. 35 shows immunofluorescence localization of RAG1AP1 in human liversections. RAG1AP1 was stained by antibody against the C-terminal peptideof human RAG1AP1 (Abcam) and Alexa fluor 594-labeled donkey-anti-goatIgG. Golgin-97 was used as golgi-marker, which was stained by monoclonalantibody against golgin-97 (Invitrogen) and Alexa fluor 488-labeleddonkey-anti-mouse IgG.

FIG. 36 shows Golgi-targeted FLIPglu-600μΔ13V. FRET glucose sensor wastargeted to golgi using peptide (14-44) of β-1,4-galactosyltransferase I(galT) and stem (Schaub et al, Mol Biol Cell, 17: 5153-5162, 2006).

FIG. 37 shows Golgi-targeted FLIPglu-600μΔ13V. FRET glucose sensor wastargeted to golgi using peptide (14-44) of β-1,4-galactosyltransferase I(galT) and stem (Schaub et al, Mol Biol Cell, 17: 5153-5162, 2006).

FIG. 38 shows sugar flux analysis in cytosolic and golgi of MDCK cellswith FRET glucose sensor. FRET analysis in MDCK cell in the absence(A,B) or presence (C,D) of expressing RAG1AP1 and co-expressing withcytosolic (A,C) or golgi targeted (B,D), FRET glucose sensorFLIPglu-600μΔ13V. Cells were perfused with different external glucoseconcentrations (1, 2.5, 5, 10, and 40 mM). FRET images were acquired anddata were analyzed. Data are mean±SD (n=4-15).

FIG. 39 shows sugar flux analysis in cytosolic and golgi of Hela cellswith FRET glucose sensor. FRET analysis in Hela cell expressingcytosolic (A) or golgi targeted (B), FRET glucose sensorFLIPglu-600μΔ13V. Cells were perfused with different external glucoseconcentrations (5, 10, and 40 mM) and galactose (5 and 40 mM). FRETimages were acquired and data were analyzed. Data are mean±SD (n=7-8).

FIG. 40 shows sugar flux analysis in HEK293T cells expressing C. elegansGLUE family members with FRET glucose sensor. FRET analysis in HEK293Tcells expressing C. elegans GLUE members coexpressing with FRET glucosesensor, FLIPglu-600μΔ13V. Cells were perfused with different externalglucose concentrations (2.5, 5, 10, and 40 mM) and galactose (5 and 40mM). FRET images were acquired and data were analyzed. Data are mean±SD(n=12-24).

FIG. 41 shows that the human RAG1AP1 homolog is a sugar effluxtransporter. Human SGLT1 sodium glucose cotransporter (“S”) which hasbeen previously shown to be a secondary active glucose importer. SGLT1is endogenously expressed in Xenopus oocytes and can mediate uptake of14C labelled glucose into the oocyte. When RAG1AP1 (“R”) is coexpressed,less uptake is seen in the oocytes. This is compatible with a glucose‘leak’ due to RAG1AP1 activity that prevents high accumulation ofglucose in the SGLT1 expressing cells.

FIG. 42 shows the 14C glucose uptake data for five of the C. eleganshomologs. At least 3, 4 and 5 are active. (code for names of C elegansgenes: Ce1: C06G8.1; Ce2: K06A4.4; Ce3: Y39A1A.8; Ce4: K11D12.5; Ce5:K02D7.5; (worm mutants of Ce5: K02D7.5 show increased fat accumulation,consistent with reduced efflux of glucose from these cells).

FIGS. 43A through 43H show sucrose exporting function of SWEETs inHEK293 cells (Positive control (potato sucrose transporter StSUT1;Riesmeier et al. 1993 Plant Cell) is 43A, negative control (empty vector43B). Uptake of sucrose was determined using the FRET sucrose sensorFLIPsuc90μΔ1 (Chaudhuri et al., 2008 Plant Journal). SWEET 10 (FIG.43C), SWEET 11 (FIG. 43D), SWEET12 (FIG. 43E), SWEET 13 (FIG. 43F),SWEET 14 (FIG. 43G), and OsSWEET11/Os8N3 (FIG. 43H) showed a negativeFRET response (negative ratio change corresponds to an increase incytosolic sugar content) similar to the one for the positive controlStSUT1 indicating sucrose uptake into the mammalian cell. This uptakecould be mediated by uniport (facilitated diffusion), proton symport orproton antiport.

FIG. 44 shows the amino acid sequence for various GLUE proteins in C.elegans, mouse, rat, human, Arabidopsis, rice, Medicago, and petunia.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Other objects, advantages and features of the present invention maybecome apparent to one skilled in the art upon reviewing thespecification and the drawings provided herein. Thus, further objectsand advantages of the present invention will be clear from thedescription that follows.

Plants require sugar efflux transporters to support seed and pollendevelopment, produce nectar and nurture beneficial microorganisms in therhizosphere. The identity of these efflux transporters however hasremained elusive. Using optical sugar sensors, a novel class of sugartransporters (“GLUE”, “Glüs”, or “SWEET”) has been identified fromplants. Arabidopsis and rice GLUEs functions as an im- and exporters ofsugars. OsGluE11/Os8N3 and AtGluE8/RPG1 are required for pollenviability. Expression of GLUE homologs in nectaries and root nodulessuggests roles in feeding pollinators and symbionts. Fungal andbacterial pathogens modulate mRNA levels of different GLUE members toco-opt mono-, di- or oligosaccharide transport activity. OsGLUE11/Os8N3functions as a host susceptibility factor for bacterial blight, linkingGLUEs to both plant and pathogen nutrition.

The human genome contains at least two classes of glucose transporters,SLC2 and SLC5. SLC2, named GLUTs are uniporters, i.e. they transportglucose along its concentration gradient. In contrast, SGLTs areNa⁺-coupled cotransporters that can actively import glucose driven by asodium gradient. These transporters can explain most of the uptakeactivities found in humans, e.g. a GLUT2 mouse knock-out mutant showsdramatically reduced uptake capacity but surprisingly not the cellularefflux. However, bioinformatic analyses showed that animals and humangenomes contain homologs of the SWEETs, registered as solute carrierfamily SLC50. The C. elegans genome contains 7 homologs of a novel classof sugar efflux transporters (SLC50), while the human genome has asingle homolog, named RAG1AP1.

Sugar efflux is an essential process required for cellular exchange ofcarbon skeletons and energy in multicellular organisms and ininteractions between organisms. Sugar efflux from the tapetum ortransmitting tract of the style fuels pollen development and later onpollen tube growth. Flowers secrete sugars for nectar production toattract pollinators and plants secrete carbohydrates into therhizosphere, potentially to feed beneficial microorganisms (T. Bisselinget al., Science 324, 691 (2009)). Sugar efflux carriers are required atmany other sites, including the mesophyll in leaves and the seed coat(Y. Zhou et al., Plant J. 49, 750 (2007)). The molecular nature of theefflux transporters is unknown (S. Lalonde et al., Annu. Rev. PlantBiol. 55, 341 (2004)). Plant-derived sugars also provide a substrate forpathogens. The primary goal of pathogens is to access nutrients from itshost plant to efficiently reproduce. Phytopathogenic bacteria in thegenera Pseudomonas and Xanthomonas can live in the extracellular space(apoplasm) of plant tissue, where they acquire carbohydrates as theirsource of energy and carbon skeletons. Successful pathogens likelyco-opt such mechanisms to alter nutrient flux (J. W. Patrick, Aust. J.Plant Physiol. 16, 53 (1989)). As a consequence, pathogens and plantsengage in an evolutionary tug-of-war in which the plant tries to limitpathogen access to nutrients and initiates defense strategies, while thepathogen devises strategies to gain access to nutrients and suppresshost immunity. Insight to the mechanisms used by pathogens to alterplant defenses is now emerging; however, little is known about howpathogens alter host physiology, notably sugar export, to supportpathogen growth. The present invention has identified the existence oftransporters, either vesicular or at the plasma membrane, that secretesugars. The present invention has further identified that these plantefflux transporters are ‘co-opted’ by pathogens to supply their nutrientrequirements (J. W. Patrick, Aust. J. Plant Physiol. 16, 53 (1989)). Atleast in the case of wheat powdery mildew, glucose is the main sugartransferred from plant host to pathogen (J. Aked, J. L. Hall, NewPhytol. 123, 271 (1993); P. N. Sutton et al., Planta 208, 426 (1999);and P. N. Sutton et al., Physiol. Plant 129, 787 (2007)). Respectivepathogen glucose/H⁺ cotransporters have been identified (R. T. Voegeleet al., Proc. Natl. Acad. Sci. USA 98, 8133 (2001)); in contrast, theplant sugar efflux mechanisms have previously remained elusive.

In many metabolic pathways, a transporter may function at either or bothends of a particular pathway to supply and remove the substrate andproduct respectively from the presence of the enzyme(s). Transport canbe via passive transport, active transport, diffusion, or osmosis.Transporters may directly or indirectly be responsible for the presenceor absence of a substrate from an enzyme. Transporters may be localizedin or near the cell membrane, or they may be located in the cytoplasm ornear or in other organelles such as the endoplasmic reticulum,mitochondria, chloroplast, peroxisomes, golgi apparatus, vesiclesnuclear membrane, or vacuole, lysosome or plasma membrane.

A transporter may be stationary and allow passage of the substrate by orthrough it, or it may bind the substrate and physically shuttle thesubstrate to a particular subcellular destination. A transporter maybind one type of molecule to allow passage or transport of another typeof molecule. The transporter may move independently or through the aidof other proteins, such as protein kinases or ATP-cleaving domains.

Transporters determine the uptake or emission of a substance into or outof a cell or an organism, and transporters control the transport anddistribution of substances between the cells. Transporters may alsofunction intercellularly, such as transporting between organelles, forexample, in and out of the nucleus. As transporters often lie at thebeginning or the end of a metabolic pathway, they thereby take charge offundamental higher controlling functions. Transporters maybe involved inthe reuptake of a released small molecule such as a monoamine orneurotransmitter.

Some transporters require energy to transport their particularsubstrate. In certain instances, the energy is supplied through ATP, anda resulting phosphorylation of the transporter causes a conformationalchange that allows the transport to proceed. In other cases, the energyis provided indirectly through coupling of the transport to a secondsubstrate, e.g., the proton or sodium/potassium gradient created by a Por V-type ATPase. In other instances, the interaction of a transporterwith another protein or molecule will cause a conformational change toallow transport of the same protein or molecule or a different proteinor molecule to proceed. In yet further instances, the separation betweenthe transporter and a regulatory protein causes a conformational changein the transporter to allow transport to proceed. A transporter mayinteract with a substrate or product through direct binding. Duringcatalysis of transport, the transporter can undergo a conformationalchange. This also includes hybrid proteins that serve as enzymes andtransporters such as P-type or V-type ATPases.

As used herein, the term “conformational change” refers to anyconformational change occurring in the sensor, such as, effects on the3D location of atoms and atom groups in the protein, the averageposition of movable atoms and side chains, changes in the surfaceproperties of the protein, movements of domains folding/unfolding ofdomains that effects either the position/average position orconformation of a single or the relative position, average position ofthe fluorophore is changed resulting in a change of energy emitted bysaid detection portions. The term “relative position” refers to anypossible kind of spatial relationship the two detection portions canhave to one another such as distance and orientation. For instance, theconformation may change by rotation of one or several atoms, side chainsor domains, by folding up the enzyme, by twisting one or both of thedomains laterally or by any combination of these movements. Useful is aconformational change where the orientation or distance between thedetection portions is altered or a change that exerts an effect on theconformation of the reporter element. Alternatively, the conformationalchange in the enzyme portion affects the properties of a single attachedfluorophore. In this case, a second fluorophore may be used to obtain aRET signal. In such cases, it is advantageous that, either beforebinding or upon binding, the detection portions are oriented in a waythat at least half-maximum energy transfer takes place.

As used herein, “ligand” refers to a molecule or a substance that canbind to a protein such as a periplasmic binding protein to form acomplex with that protein. The binding of the ligand to the protein maydistort or change the shape of the protein, particularly the tertiaryand quaternary structures. A ligand maybe a substrate. A substrate mayinclude an educt, or a reagent which is converted to a product throughthe assisted catalysis of the enzyme. A ligand may be an analog orderivative of an endogenous ligand. A ligand may compete with anendogenous ligand for the binding site. The ligand may be, for example,a small molecule, a chemical, a single stranded oligonucleotide, adouble-stranded oligonucleotide, DNA, RNA, or a polypeptide. The ligandmay be a transition analog or a product. The ligand includes anychemical bound to the protein, including an ion such as magnesium or anallosteric factor or another protein. The ligand may be a sugar, such asglucose.

As used herein, “fluorescent indicator” refers to a fluorescent domainor compound linked to the PBP. Changes in the shape of the PBP result inchanges of the fluorescence of the fluorescent domain or compound,thereby indicating the change of shape in the enzyme. The domain may bea fluorescent protein. The fluorescent domain may comprise twosubdomains, such as a donor and an acceptor fluorophore. In someinstances, the PBP will be covalently linked in between the donor andacceptor fluorophores. Alternatives to the use of fluorescent indicatorsare luminescent or phosphorescent molecules, as well as compounds thatmay be detected by other means such as NMR, polarization detectors, etc.

As used herein with respect to proteins and polypeptides, the term“recombinant” may include proteins and/or polypeptides and/or peptidesthat are produced or derived by genetic engineering, for example bytranslation in a cell of non-native nucleic acid or that are assembledby artificial means or mechanisms.

As used herein, “fusion” may refer to nucleic acids and polypeptidesthat comprise sequences that are not found naturally associated witheach other in the order or context in which they are placed according tothe present invention. A fusion nucleic acid or polypeptide does notnecessarily comprise the natural sequence of the nucleic acid orpolypeptide in its entirety. Fusion proteins have the two or moresegments joined together through normal peptide bonds. Fusion nucleicacids have the two or more segments joined together through normalphosphodiester bonds.

As used herein, the term “biological sample” refers to a collection ofcells or cellular matter. The sample may be obtained from an organism orfrom components (e.g., cells) of an organism. The sample may be obtainedfrom any biological tissue or fluid. The sample may be a sample which isderived from a subject. The subject may be a plant. The sample may beobtained from a plant or a component of a plant. The subject may be ananimal. The animal may be a mammal, such as a human or a human patient.Such samples include, but are not limited to, sputum, blood, blood cells(e.g., white cells and red cells), tissue or biopsy samples (e.g., tumorbiopsy), urine, peritoneal fluid, and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues such as frozensections taken for histological purposes. Biological samples may alsoinclude in vitro cell cultures. Cell cultures may be immortalized celllines or primary cell lines. Cell cultures may include different celltypes.

As used herein, the term “dsRNA” refers to double-stranded RNA, whereinthe dsRNA may be double-stranded by two separate strands or by a singlestranded hairpin. dsRNA may comprise a nucleotide sequence homologous tothe nucleotide of a target gene. dsRNA may be produced by expressionvectors (also referred to as RNAi expression vectors) capable of givingrise to transcripts which form self-complementary dsRNAs, such ashairpin RNAs or dsRNA formed by separate complementary RNA strands incells, and/or transcripts which can produce siRNAs in vivo. Vectors mayinclude a transcriptional unit comprising an assembly of (1) geneticelement(s) having a regulatory role in gene expression, for example,promoters, operators, or enhancers, operatively linked to (2) a “coding”sequence which is transcribed to produce a double-stranded RNA (two RNAmoieties that anneal in the cell to form an siRNA, or a single hairpinRNA which can be processed to an siRNA), and (3) appropriatetranscription initiation and termination sequences. The choice ofpromoter and other regulatory elements generally varies according to theintended host cell. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome.

As used herein, the term “isolated” refers to molecules separated fromother cell/tissue constituents (e.g. DNA or RNA), that are present inthe natural source of the macromolecule. The term “isolated” as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, and culture medium whenproduced by recombinant DNA techniques, or that is substantially free ofchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” may include nucleic acid fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state.

As used herein, the term “multimer” refers to formation of a multimericcomplex between two or more distinct molecules. The multimer complex maycomprise, for example, two or more molecules of the same protein (e.g.,a homo-dimer, -trimer, -tetramer, dimer of dimers or higher ordermultimer) or a mixture of two or more different (i.e., non-identical)proteins (e.g. a hetero-dimer, dimer of different dimers, -trimer,-tetramer or higher multimer). For example, multimeric antibodies maycomprise the same antibody or two or more different antibodies, each ofwhich have two or more functions or activities (e.g., bind to two ormore epitopes).

As used herein, “subject” may include a recipient of the invention. Thesubject can be a plant, or a component of a plant, such as a plant organor organelle. The subject can be any animal, including a vertebrate. Thesubject will in most cases, preferably be a human, but may also be adomestic livestock, laboratory animal (including but not limited to,rodents such as a rat or mouse) or pet animal.

As used herein, the term “variant” refers to polypeptides with at leastabout 70%, more preferably at least 75% identity, including at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to nativemolecules by BLAST analysis. Many such variants are known in the art, orcan be readily prepared by random or directed mutagenesis of a nativefluorescent molecules (see, for example, Fradkov et al., FEBS Lett.479:127-130 (2000).

As used herein, the term, “plasmid” and “vector” are usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto. A vector may be any of a number ofnucleic acids into which a desired sequence may be inserted byrestriction and ligation for transport between different geneticenvironments or for expression in a host cell. Vectors are typicallycomposed of DNA, although RNA vectors are also available. Vectorsinclude, but are not limited to, plasmids and phagemids. A cloningvector is one which is able to replicate in a host cell, and which isfurther characterized by one or more endonuclease restriction sites atwhich the vector may be cut in a determinable fashion and into which adesired DNA sequence may be ligated such that the new recombinant vectorretains its ability to replicate in the host cell. In the case ofplasmids, replication of the desired sequence may occur many times asthe plasmid increases in copy number within the host bacterium or just asingle time per host before the host reproduces by mitosis. In the caseof phage, replication may occur actively during a lytic phase orpassively during a lysogenic phase.

Vectors may further contain a promoter sequence. A promoter may includean untranslated nucleic acid sequence usually located upstream of thecoding region that contains the site for initiating transcription of thenucleic acid. The promoter region may also include other elements thatact as regulators of gene expression. In further embodiments of theinvention, the expression vector contains an additional region to aid inselection of cells that have the expression vector incorporated. Thepromoter sequence is often bounded (inclusively) at its 3′ terminus bythe transcription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Activation of promoters may be specific to certaincells or tissues, for example by transcription factors only expressed incertain tissues, or the promoter, may be ubiquitous and capable ofexpression in most cells or tissues.

Vectors may further contain one or more marker sequences suitable foruse in the identification and selection of cells which have beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Vectors may be those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

An expression vector is one into which a desired nucleic acid sequencemay be inserted by restriction and ligation such that it is operablyjoined or operably linked to regulatory sequences and may be expressedas an RNA transcript. Expression refers to the transcription and/ortranslation of an endogenous gene, transgene or coding region in a cell.

A coding sequence and regulatory sequences are operably joined when theyare covalently linked in such a way as to place the expression ortranscription of the coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences betranslated into a functional protein, two DNA sequences are said to beoperably joined if induction of a promoter in the 5′ regulatorysequences results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequences, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a promoter regionwould be operably joined to a coding sequence if the promoter regionwere capable of effecting transcription of that DNA sequence such thatthe resulting transcript might be translated into the desired protein orpolypeptide.

Some aspects of the present invention include the transformation and/ortransfection of nucleic acids. Transformation is the introduction ofexogenous or heterologous nucleic acid to the interior of a prokaryoticcell. Transfection is the introduction of exogenous or heterologousnucleic acid to the interior of a eukaryotic cell. The transforming ortransfecting nucleic acid may or may not be integrated (covalentlylinked) into chromosomal DNA making up the genome of the cell. Inprokaryotes, for example, the transforming nucleic acid may bemaintained on an episomal element such as a plasmid or viral vector.With respect to eukaryotic cells, a stably transfected cell is one inwhich the transfecting nucleic acid has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transfected nucleic acid.

As used herein, the term “fusion protein” or “chimeric protein” is usedto refer to a polypeptide comprising at least two polypeptides fusedtogether either directly or with the use of spacer amino acids. Thefused polypeptides may serve collaborative or opposing roles in theoverall function of the fusion protein.

As used herein, “fragments” of antibodies include but are not limited toFc, Fab, Fab′, F(ab′)₂ and single chain immunoglobulins.

As used herein, the term an “immunologically effective amount” meansthat the administration of that amount to a subject, either in a singledose or as part of a series, is effective for treatment of a disease ordisorder. This amount varies depending upon the health and physicalcondition of the subject to be treated, the species of the subject to betreated (e.g. non-human mammal, primate, etc.), the capacity of thesubject's immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

As used herein, “pharmaceutical composition” or “formulation” refers toa composition comprising an agent or compound together with apharmaceutically acceptable carrier or diluent. A pharmaceuticallyacceptable carrier includes, but is not limited to, physiologicalsaline, ringers, phosphate buffered saline, and other carriers known inthe art. Pharmaceutical compositions may also include stabilizers,anti-oxidants, colorants, and diluents. Pharmaceutically acceptablecarriers and additives are chosen such that side effects from thepharmaceutical agent are minimized and the performance of the agent isnot canceled or inhibited to such an extent that treatment isineffective.

As used herein, “therapeutically effective amount” refers to that amountof the agent or compound which, when administered to a subject in needthereof, is sufficient to effect treatment. The amount of antibodiessuch as cross-linked Aβ oligomer reactive antibodies which constitutes a“therapeutically effective amount” will vary depending on the severityof the condition or disease, and the age and body weight of the subjectto be treated, but can be determined routinely by one of ordinary skillin the art having regard to his/her own knowledge and to thisdisclosure.

A “cofactor” refers to an element that interacts with a protein toassist that protein in executing its physiological function. A cofactormay catalyze a reaction. A cofactor may associate with a protein, forexample a transporter or an enzyme, either through strong interactions,or through a loose association. A cofactor may be a “coenzyme” or a“prosthetic group.” A “coenzyme” refers to organic molecules thatshuttle or carry chemical groups between enzymes. A “prosthetic group”refers to a cofactor that binds an enzyme to become a part of theenzyme. A cofactor may also be a metal ion, such as calcium, magnesium,manganese, iron, potassium, sodium, aluminum, copper, nickel, selenium,molybdenum, or zinc. The limited supply of a cofactor may be arate-limiting element.

The term “biological fluid” includes any bodily fluid that containscirculating proteins, including plasma, serum and whole blood, saliva,cerbrospinal fluid, amniotic fluid, synovial fluid, aqueous humour,bile, cerumen, Cowper's fluid, chyle, chyme, female ejaculate andvaginal lubrication, interstitial fluid, lymph fluid, menses, mucus,pleural fluid, pus, sebum, semen, sweat, tears, vomit, urine, lactationfluids and other secretions. A protein-containing extract of abiological fluid is any preparation that is collected or separated froma biological fluid, such as immunoglobulin fractions. Blood, serum orplasma that may be used in the present invention may be freshly obtainedfrom an individual, or it may be obtained from such sources as pooledblood or plasma preparations obtained from blood banks or other bloodcollection facilities. For the purposes of the present invention, theblood, serum or plasma may also be from collections that are out-of-dateor otherwise found to be substandard by blood banks or blood collectionfacilities. Identical process of this invention can be applied to animalblood and should result in obtaining analogous animal antibodies forpurposes relating to veterinary medicine. Fluids may be used in theirwhole state as it is obtained, or may be further processed such asthrough allowing sedimentation or by centrifugation. The fluid may befrom a plant, such as a sap, phloem sap, zylem sap, nectar, resin,latex, or oil. The fluid may be a supernatant, a collected sediment, ora pellet obtained by extra-gravitational forces, such as centrifugationor filtration.

The present invention is based in part on the discovery of a novel classof sugar transporter proteins. The sugar may be a mono-, di-, oroligosaccharide, such as glucose, fructose, ribose, lactose, galactose,arabinose, maltose, amylose, cellulose, or sucrose. The transporters maytransport sugars across a membrane in a cell. The membrane may be aplasma membrane or a cell wall. The membrane may surround the cytoplasmof a cell. The membrane may surround a cellular organelle, such as amitochondrium, an endoplasmic reticulum, a golgi apparatus, a nucleus,an endosome, or a vacuole. The transporter may transport sugars betweenthe membranes of one cell/orhganelle, and the membrane of anothercell/organelle.

The present invention may provide for methods of transporting sugar inan organ or in between organs of a subject. The organ may be involved inthe processing, importation, or exportation of carbohydrates, such assugar and glucose. The organ may be involved in the digestive system.For example, the organ may be an intestine (large or small), a stomach,or a liver. The transporters of the present invention may assist intransporting sugar in or out of a cell with an organ. The transportermay work collectively with other proteins known to operate in movingsugars, such as SGLT, GLUT1, and GLUT2. The transporters may effluxsugar out of an organelle. The transporters may efflux sugar passivelythrough the formation of vesciles. For example, expression of thetransporters in a golgi apparatus may allow for efflux of sugar intoforming vesciles that then passively migrate their contents out of thecell.

Originally it was thought that the glucose uniporter GLUT2 isresponsible both for import and efflux of glucose in liver andintestine. However, knock down of GLUT2 in hepatocytes and in transgenicmice showed that GLUT2 is essential for glucose uptake but not forglucose efflux. Oral glucose load of GLUT2 knock out mice resulted innormal rates of glucose appearance in the blood (Thorens et al. J. Biol.Chem. 275, 23751-23758, (2000)). Similarly, persons affected by withFanconi-Bickel syndrome, a syndrome caused by inactivation in both GLUT2alleles (Santer et al. Nat. Genet. 17, 324-326, (1997)), did not lead toabnormal carbohydrate ingestion, a process that requires efflux fromintestinal cells (Manz, F. et al. Pediatr Nephrol 1, 509-518, (1987)).Based on its function, HsSWEET1/RAG1AP1 may assist in efflux of sugar,such as glucose, from liver.

The transporter, for example a GLUE transporter, may be located in amembrane. The transporter may span a membrane. The transporter maycomprise a pore through the membrane. Sugar transportation may bethrough a pore in the membrane created by the transporter. The pore maybe capable of varying in width in response to stimuli, thereby alteringthe ability of sugar to pass through the pore. The transporter may begated. The gating mechanism will allow passage of sugar through thetransporter in response to a stimulus. The gating mechanism may requirea ligand to bind or may involve a voltage sensor. The transporter mayallow passage of a sugar by passive diffusion. The transporter mayrequire energy, such as adenosine triphosphate, in order to transport asugar. The transporter may transport sugar from high to lowconcentration in an attempt to reach an equilibrium. The transporter maytransport sugar from an area of low concentration to a higherconcentration, thereby increasing a gradient. The transporter may be auniporter. A uniporter, as used herein, refers to a transporter thatfunctions as a facilitator and the direction of transporter isdetermined on the gradient or concentration differential across themembrane of the substrate being transported. A uniporter refers to atransporter that is able to operate self-sufficiently, without relyingon a cofactor such as a cotransported molecule or an activatingmolecule. A uniporter will typically allow flow of a sugar in thedirection of a concentration gradient, i.e., from a side with a highconcentration of sugar to a side with a low concentration of sugar.

The transporter, for example a GLUE transporter, may increase sugarconcentration on one side of a membrane. The transporter may increasesugar levels inside a cell. This could be achieved through coupling ofthe transport to the transport of second compound, which can be an ionor another metabolite, such as a proton, hydroxy-anion, sodium orpotassium. Coupling may be by cotransport or antiport or by a ping-pongmechanism. The transporter may decrease sugar concentrations within acell. The transporter may export sugar from a cell. The transporter mayimport sugar into a cell.

The transporter may further be affected by a signal, such as a kinase, asecond messenger, an anion, a cation, or a ligand. The transporter maybe affected by a cofactor. The cofactor may be an ion, such as ionizedforms of magnesium, zinc, iron, copper, iodine, chloride, sodium,potassium, calcium, manganese, sulfate, sulfate, ammonium, nitrate,nitrite, carbonate, carboxylic acid, or phosphate. The cofactor may benecessary to assist substrate or ligand binding to the transporter or toa second messenger. The cofactor may be necessary for the activity ofthe transporter. In some instances, failing to add a cofactor willprovide a non-functioning or lesser-functioning transporter. In otherinstances, the presence of a cofactor will down-regulate the activity ofthe transporter. The transporter may be downregulated throughinternalization, such as through the clathrin internalization mechanism.

The transporter may be in a cell or extract obtained from a cell. Thecell may be in a prokaryote. The cell may be in an eukaryote.

The cell may be in an animal or a rt thereof. The transporter may play arole in transporting glucose in the endoplasmic reticulum, golgiapparatus, vesicles or plasma membrane of an animal cell. The cell maybe an animal cell that is involved in glucose transport or secretion.The cell may be a glandular cell. The glandular cell may be an alveolarcell of the mammary gland.

The cell may be in a plant or a part thereof, such as a root, stem,leaf, seed, flower, fruit, anther, nectary, ovary, petal, tapetum,xylem, or phloem. By way of example, plants include embryophytes,bryophytes, spermatophyes, nematophytes, tracheophytes, soybean, rice,tomato, alfalfa, potato, pea, grasses, herbs, trees, algae, mosses,fungi, vines, ferns, bushes, barley, wheat, hops, maize, lettuce,orange, peach, citrus, lemon, lime, coconut, palm, pine, oak, cedar,mango, pineapple, rhubarb, strawberry, blackberry, blackcurrant,blueberry, raspberry, kiwi, grape, rutabega, parsnip, sweet potato,turnip, mushroom (Fungus), pepper, cilantro, onion, leek, fennel, clove,avocado, or cucumber. It also includes biofuel crops such as Miscanthusor switchgrass, poplar, Sorghum, and Brachypodium.

The transporters of the present invention, for example GLUEtransporters, may transport sugar, specifically mon-, di- oroligosaccharides e.g. glucose or sucrose, within an organism, such as aplant or animal. In plants, the transporters may transport sugars forthe production of nectar. The transporters may transport sugar to and/orfrom the nectaries of a plant. The transporters of the present inventionmay be localized to the nectaries. As used herein, a “nectary” refers toa secretory structure that produces nectar. Nectar is a compositioncomprising glucose and/or fructose and/or other saccharides which mayserve as a reward for pollinators.

The transporters may transport sugar to and/or from the anther of aplant. The anther refers to a reproductive organ of a plant, comprisedof a stamen and a filament. The transporters of the present inventionmay be localized to the anther. The transporters of the presentinvention may be localized to the stamen and/or filament of the anther.They may localize to the tapetum or the pollen itself. In the pollenthey may localize to the vegetative or generative cells. The presence ofthe transporters of the present invention may affect the functioning ofthe anther. The functioning of the transporters in the tapetum may playa role in pollen nutrition. The functioning in the pollen may helpnourish the generative cells. The functioning in the anther may cause asudden hydrolysis of starch, which may lead to an increase in theosmotic potential, which in turn may lead to retraction of water fromsurrounding tissues, which may then promote dehydration and dehiscenceof the anther. The functioning may also contribute to uptake of sugarsinto the pollen or release of sugar in the transmitting tract orepididymis.

The transporters of the present invention may transport sugar to and/orfrom the sporangium of a plant, such as a microsporangium or amegasporangium. The transporters of the present invention may belocalized to a sporangium or spore releasing reproductive gland. Thetransporters may affect the function of a sporangium.

The transporters of the present invention may transport sugar to and/orfrom the transmission tract to supply the elongating pollen tube withnutrients and energy.

The transporters of the present invention may mediate uptake across theplasma membrane and ‘efflux’ into the ER. The transporters of thepresent invention may function as a glucose uniporter, for which thedirection of transport depends only on the glucose gradient across themembrane.

The transporters, such as a GLUE transporter, may induce development ofthe phloem cells. The transporters may be localized to the phloem. Thetransporters may affect the function of a phloem cell. The phloem refersto tissue involved in transporting nutrients, such as sap, in a cell.The phloem may transport nutrients from a certain region, such as a rootor a sugar source of a plant, to another region of a plant, such as aleaf or a sugar sink of a plant. Transport along the phloem may bemulti-directional or unidirectional. The phloem may comprise parenchymacells, sieve-tube cells, mesophyll cells and companion cells, such asordinary companion cells, transfer cells and intermediary cells. Thephloem may further comprise albuminous cells, fibers and sclereids.

The transporters of the present invention may affect thedisease-susceptibility of an organism, such as a plant or animal. Thetransporters of the present invention may affect the susceptibility of aplant to a pathogen, such as a virus or bacteria or insect. It is knownthat pathogens may affect gene transcription of a host cell. Thepathogen may affect gene transcription of sugar transporters. Thepresent invention provides for novel methods to protect the host cell.They may affect the nutrition of both symbionts and pathogens above andbelow ground. They may attract microorganisms. They may play a role insecreting sugars into soil. They thus may affect the microflora aroundthe root as well as the productivity of the plant. They may affect theinteraction with pollinators. They may play a role in supplying sugarsto cells in the plant that depend on external supply, such as epidermis,guard cells, seeds.

Transporter Proteins

The present invention provides for a continuous sequence of polypeptidesthat collectively function in the passage of sugar across a membrane.The transporters may be imbedded in or completely traverse a membrane.The transporter may traverse a membrane multiple times, such as 2, 3, 4,5, 6, 7, or more times. Those skilled in the art will appreciate thatthe portions of a transporter that cross a membrane will vary inhydrophobicity and hydrophilicity as compared with those portions of thetransporter positioned on the exterior (either side, such asextracellular and intracellular) of the membrane. The transporters maycomprise at least 2 subunits, such as two transmembrane proteins, forexample, a homo or heterotrimer, wherein the term “trimer” refers to thenumber of times the protein spans across a membrane. In furtherinstances the subunits may be connected by a linker peptide, such as afurther intracellular domain, a further extracellular domain or afurther transmembrane domain.

The transporters may form a pore. The pore may be formed by a sphericalarrangement of the transmembrane domains of the transporter or thesub-domains thereof. The pore may allow passage of a sugar through it.The pore may be selective for passage of sugar only. The pore may have aselective point or points which restrict passage to certain sized orcertain shaped molecules. Passage through the pore may be based on aconcentration gradient only. The pore may further be opened or closedbased on the activity of a cofactor, such as activity of an interactingprotein, or the binding of an ion or the presence of a charge, such as anegative or positive charge.

The present invention also provides chimeric transporters. Chimeric area combination of functional domains derived from two or, more differentproteins. Chimeric transporters may fuse the pore of the transporter toa second messenger interacting/recruiting domain from another membraneassociated protein, such as a receptor tyrosine kinase, a G-proteincoupled receptor, an aquaporin, or another transporter. The chimerictransporter may be a fusion of two or more of the transporters describedherein. By way of example, transporters may include, glucosetransporters, glutamate transporters (sodium dependent and vesicular),aquaporins, Na/K ATPase, serotonin transporter (SERT), dopaminetransporter (DAT), norepinephrine transporter (NET), ammoniumtransporters, and potassium channels.

The present invention also provides fusion proteins of the transporters.For example, a known epitope or tag may be fused to the transporter. Thetag may be a fluorescent tag, such as a green fluorescent protein, redfluorescent protein, orange fluorescent protein, yellow fluorescentprotein, cyan fluorescent protein, or blue fluorescent protein. Methodsfor preparing fusion proteins are known in the art.

The present invention provides for transporters that may provide fortransport of a sugar. The transport may be across a membrane. Thetransport may be exporting a sugar from a cell or from an organellewithin a cell. The transport may be importing a sugar into a cell or anorganelle within a cell. The sugar may be a mono-, di-, oroligo-saccharide or derivative thereof. For example, sugars may includeribose, arabinose, a pentose, such as glucose, fructose, galactose, andmannose, hexose, such as maltose and sucrose. Di-sacchrides may includeraffinose and stachyose. Derivates may include glycosyl-derivatives ofamino acids and hormones

The present invention also provides nucleic acids encoding thetransporters of the present invention, such as GLUE transporters. Thepresent invention discloses several cDNAs that encode the transportersof the present invention. The protein MtN3 from Medicago and homologsthereof may function as a GLUE. In Arabidopsis, the following AccessionNos: encode transporters: AT4G15920, AT3G16690, AT5G13170, AtSAG29,AT4G25010, AT5G50800, AT5G23660, AT3G48740, AT5G50790, AT2G39060,AT5G40260, AtRPG1, AT4G10850, AT1G66770, AT5G62850, AtVEX1, AT3G28007,AT3G14770, AT1G21460, and AT5G53190 (all of which are hereinincorporated by reference in their entirety). In Petunia plants, NEC1 isan example of a sugar transporter (which is herein incorporated byreference in their entirety). In Medicago plants, the following cDNAAccession Nos that encode the transporters of the present invention havebeen identified: AC202585, AC147714, MtC60432 GC, MtC11004 GC, CT963079,MtD03138 GC, TC 125536, AC146866, AC189276, TC129646, CAA69976 MtNod3AC2456, TC115479, AC146747, MtC10424 GC, CT954252, CU302340, AC202585,AC147714, MtC60432 GC, MtC11004 GC, and CT963079 (all of which areherein incorporated by reference in their entirety). In rice plants, thefollowing cDNA Accession Nos that encode the transporters of the presentinvention have been identified: Os08g42350 (Os8N3) Os08g0535200,Os12g29220 Os03g0347500, Os05g51090 Os05g0588500, Os12g07860,Os09g08440, Os09g08490, Os09g08270, Os09g08030 Os09g0254600,Os01g42090.1 Os01g0605700, Os01g42110.1 Os01g060600, Os02g19820Os02g03110, Os05g35140 Os05g0426000, Os01g65880 Os01g0881300, Os01g50460Os01g0700100,) s01g36070.1 Os01g0541800, Os01g12130.1, Os05g12320Os05g0214300, and Os01g21230 (all of which are herein incorporated byreference in their entirety).

The invention also comprises the animal homologs RAG1AP1 as well thebacterial homologs of this family such as those encoded by the followingaccession nos: A1BJ76 (SEQ ID NO: 91), A1VHH8 (SEQ ID NO: 92), A3IH65(SEQ ID NO: 93), A4AVY5 (SEQ ID NO: 94), A5ERR3 (SEQ ID NO: 95), A5FEJ3(SEQ ID NO: 96), A5G4U0 (SEQ ID NO: 97), A5IEV6 (SEQ ID NO: 98), A8AYJ9(SEQ ID NO: 99), B0SHL1 (SEQ ID NO: 100), B0SR19 (SEQ ID NO: 101),B1MYL5 (SEQ ID NO: 102), B1MZF9 (SEQ ID NO: 103), B1WTC6 (SEQ ID NO:104), B3EHG6 (SEQ ID NO: 105), B5EHF6 (SEQ ID NO: 106), B5YGD6 (SEQ IDNO: 107), B6IU72 (SEQ ID NO: 108), Q11VQ0 (SEQ ID NO: 109), Q39VX0 (SEQID NO: 110), Q3B6J0 (SEQ ID NO: 111), Q5WTV4 (SEQ ID NO: 112), Q5X228(SEQ ID NO: 113), Q72RB5 (SEQ ID NO: 114), Q72FY5 (SEQ ID NO: 115),Q89G85 (SEQ ID NO: 116), Q8F4F7 (SEQ ID NO: 117) (all of which areherein incorporated by reference in their entirety). GLUEs may beobtained from prokaryotes such as Legionella, Desulfovibrio,Bradyrhizobium, Leptospira, Rhodopseudomonas, Streptococcus, Geobacter,Pelodictyon, Cytophaga, Rhodospirillum, Thermodesulfovibrio, Chlorobium,Wolbachia, Cyanothece, Leuconostoc, and Flavobacterium.

The transport of sugars is essential. For example, a glucose efflux isneeded at many points in the body of an organism, for example in thedevelopment of pollen or in the role of the epididymis feedingdeveloping sperm cells. GLUEs may be upregulated during certainphysiological processes, such as during lactation, and may be localizedto the glandular cells that secrete lactose into the milk duct.Similarly, organs such as the liver, needs to efflux glucose to keepblood glucose levels constant. The GLUEs may be involved in loadingvesicles or the Golgi with glucose for a vesicular efflux pathway.

The members of the transporter families share substantial identity.GLUE1 is 41% identical to its paralog GLUE8, and belongs to the secondof the four Arabidopsis GLUE clades. Mutation of GLUE8/RPG1 had beenshown to lead to male sterility. Coexpression of GLUE88/RPG1 with theFRET sensors for glucose in mammalian cells evidence that some GLUEs,such as GLUE8, also function as uniporters. Moreover GLUE8/RPG1complements the yeast glucose transport mutant. GLUE8/RPG1 may beexpressed in the tapetum, demonstrating a role in pollen nutrition.

GLUE1 and GLUE8 share 34% amino acid sequence identity with the riceprotein OsGLUE11/Os8N3 (named OsGLUE11 based on phylogeny). The closestArabidopsis homolog shares 40% identity with OsGLUE11/Os8N3 and belongsto the third GLUE clade. Similar to GLUE8, OsGLUE11/Os8N3 may functionin pollen nutrition since a reduction of its expression byRNA-inhibition led to reduced starch content in pollen as well as pollensterility. Silencing of Petunia Nec1, another homolog of GLUEs in clade3 also may lead to male sterility. Nec1 is expressed in nectaries, andits developmental regulation correlated inversely with starch content ofthe nectaries, demonstrating a second role for Nec1 in sugar secretionin nectaries.

The present invention provides for transporters in other organisms. Forexample, the C. elegans genome contains 7 homologs of a novel class ofsugar efflux transporters (SLC50), while the human genome has a singlehomolog, named RAG1AP1 (or HsGLUE1). Similar to the Arabidopsis GLUE1,C. elegans CeGLUE1 may mediate glucose uptake. CeGLUE1 as well as humanRAG1AP1, may counteract secondary active glucose accumulation mediatedby the Na⁺/glucose cotransporter SGLT1. Mutation of CeGLUE1 as well ashuman RAG1AP1, may lead to fat accumulation, compatible with a defect incellular glucose efflux leading to accumulation of lipids.

The present invention provides nucleic acids encoding the sugartransporters, such as GLUE. The present invention also provides nucleicacids that encode polypeptides with conservative amino acidsubstitutions. The nucleic acids of the present invention may encodepolypeptides that transport sugar. The isolated nucleic acids may haveat least about 30%, 40%, 50%, 60%, 70%, 80% 85%, 90%, 95%, or 99%sequence identity with the above identified sequences. The isolatednucleic acids may encode a polypeptide having an amino acid sequencehaving at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or99% sequence identity to amino acid sequences encoded by the aboveidentified accession numbers. The isolated nucleic acid encoding atransporter may hybridize to the above identified nucleic acidsequences.

The proteins of the GLUE share sequence and subdomain homology. Table 1below provides an illustration of the amount of sequence conservationacross a selection of GLUEs and Table 2 below provides a comparison ofGLUEs by sequence.

TABLE 1 Pairwise comparison of GLUEs by identity GLUE1 GLUE2 GLUE3GLUE10 GLUE15 GLUE11 GLUE12 GLUE13 GLUE14 GLUE1 40 40 33 33 35 35 37 36GLUE2 37 31 29 31 34 33 33 GLUE3 30 30 30 31 31 31 GLUE10 45 44 44 46 44GLUE15 46 49 45 44 GLUE11 86 56 55 GLUE12 58 57 GLUE13 75 GLUE14 GLUE9GLUE16 GLUE17 GLUE4 GLUE5 GLUE6 GLUE7 GLUE8 GLUE9 GLUE16 GLUE17 GLUE4GLUE5 GLUE6 GLUE7 GLUE8 GLUE1 31 36 38 39 40 39 42 41 GLUE2 32 33 32 3230 34 33 31 GLUE3 31 36 34 35 32 33 34 34 GLUE10 44 33 34 32 31 30 32 31GLUE15 44 35 35 34 33 30 32 34 GLUE11 47 36 36 32 30 30 32 34 GLUE12 4735 35 34 31 32 32 35 GLUE13 45 32 33 30 30 28 30 33 GLUE14 45 32 33 3131 28 30 33 GLUE9 33 36 35 30 35 36 32 GLUE16 72 40 35 34 38 38 GLUE1739 37 35 38 37 GLUE4 58 48 47 44 GLUE5 46 49 44 GLUE6 74 40 GLUE7 43GLUE8

TABLE 2 Pairwise comparison of GLUEs by sequence GLUE1 GLUE2 GLUE3GLUE10 GLUE15 GLUE11 GLUE12 GLUE13 GLUE14 GLUE1 58 57 54 54 53 53 55 53GLUE2 54 52 52 53 54 56 54 GLUE3 55 54 51 54 51 54 GLUE10 65 65 65 66 64GLUE15 66 68 64 64 GLUE11 92 71 71 GLUE12 74 74 GLUE13 86 GLUE14 GLUE9GLUE16 GLUE17 GLUE4 GLUE5 GLUE6 GLUE7 GLUE9 GLUE16 GLUE17 GLUE4 GLUE5GLUE6 GLUE7 GLUE8 GLUE1 50 54 55 55 56 61 61 58 GLUE2 58 54 54 56 56 5656 56 GLUE3 51 58 56 56 55 53 54 57 GLUE10 69 56 56 58 56 53 56 57GLUE15 69 62 59 57 58 52 56 60 GLUE11 69 60 61 52 56 50 51 52 GLUE12 6859 60 53 58 53 54 52 GLUE13 66 57 57 51 54 52 53 53 GLUE14 64 55 55 5356 54 54 53 GLUE9 61 60 55 55 56 58 57 GLUE16 84 58 55 54 56 57 GLUE1756 56 54 57 57 GLUE4 74 64 64 60 GLUE5 68 70 61 GLUE6 86 62 GLUE7 63

The nucleic acid encoding the GLUE proteins may be genetically fused toexpression control sequences for expression. Suitable expression controlsequences include promoters that are applicable in the target hostorganism. Such promoters are well known to the person skilled in the artfor diverse hosts from prokaryotic and eukaryotic organisms and aredescribed in the literature. For example, such promoters may be isolatedfrom naturally occurring genes or may be synthetic or chimericpromoters. Likewise, the promoter may already be present in the targetgenome and may be linked to the nucleic acid molecule by a suitabletechnique known in the art, such as for example homologousrecombination.

The present invention also provides expression cassettes for insertingthe nucleic acid encoding a GLUE into target nucleic acid molecules suchas vectors or genomic DNA. For this purpose, the expression cassette isprovided with nucleotide sequences at the 5′- and 3′-flanks tofacilitate removal from and insertion into specific sequence positionslike, for instance, restriction enzyme recognition sites or targetsequences for homologous recombination as, e.g. catalyzed byrecombinases.

The present invention also relates to vectors, particularly plasmids,cosmids, viruses and bacteriophages used conventionally in geneticengineering, that comprise a nucleic acid molecule or an expressioncassette of the invention.

In a preferred embodiment of the invention, the vectors of the inventionare suitable for the transformation of fungal cells, plant cells, cellsof microorganisms (i.e. bacteria, protists, yeasts, algae etc.) oranimal cells, in particular mammalian cells. Preferably, such vectorsare suitable for the transformation of human cells. Methods which arewell known to those skilled in the art can be used to constructrecombinant vectors; see, for example, the techniques described inSambrook and Russell, Molecular Cloning: A Laboratory Manual, CSH Press,2001, and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y., 1989. Alternatively,the vectors may be liposomes into which the nucleic acid molecules orexpression cassettes of the invention can be reconstituted for deliveryto target cells. Likewise, the term “vector” refers to complexescontaining such nucleic acid molecules or expression cassettes whichfurthermore comprise compounds that are known to facilitate genetransfer into cells such as polycations, cationic peptides and the like.

In addition to the nucleic acid molecule or expression cassette of theinvention, the vector may contain further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions. Generally, the vector also contains one ormore origins of replication. The vectors may also comprise terminatorsequences to limit the length of transcription beyond the nucleic acidencoding the transporters of the present invention.

Advantageously, the nucleic acid molecules contained in the vectors areoperably linked to expression control sequences allowing expression,i.e. ensuring transcription and synthesis of a translatable RNA, inprokaryotic or eukaryotic cells.

For genetic engineering, e.g. in prokaryotic cells, the nucleic acidmolecules of the invention or parts of these molecules can be introducedinto plasmids which permit mutagenesis or sequence modification byrecombination of DNA sequences. Standard methods (see Sambrook andRussell, Molecular Cloning: A Laboratory Manual, CSH Press, 2001) allowbase exchanges to be performed or natural or synthetic sequences to beadded. DNA fragments can be connected to each other by applying adaptersand linkers to the fragments. Moreover, engineering measures whichprovide suitable restriction sites or remove surplus DNA or restrictionsites can be used. In those cases, in which insertions, deletions orsubstitutions are possible, in vitro mutagenesis, “primer repair”,restriction or ligation can be used. Sequence analysis, restrictionanalysis and other methods of biochemistry and molecular biology arecarried out as analysis methods.

The present invention also provides for directed expression of nucleicacids encoding the transporters. It is known in the art that expressionof a gene can be regulated through the presence of a particular promoterupstream (5′) of the coding nucleotide sequence. Tissue specificpromoters for directing expression in a particular tissue in an animalare known in the art. For example, databases collect and share thesepromoters (Chen et al., Nucleic Acids Res. 34: D104-D107, 2006). Inplants, promoters that direct expression in the roots, seeds, or fruitsare known.

The present invention further provides isolated polypeptides comprisingtransporters fused to additional polypeptides. The additionalpolypeptides may be fragments of a larger polypeptide. In oneembodiment, there are one, two, three, four, or more additionalpolypeptides fused to the transporter. In some embodiments, theadditional polypeptides are fused toward the amino terminus of thetransporter. In other embodiments, the additional polypeptides are fusedtoward the carboxyl terminus of the transporter. In further embodiments,the additional polypeptides flank the transporter. In some embodiments,the nucleic acid molecules encode a fusion protein comprising nucleicacids fused to the nucleic acid encoding the transporter. The fusednucleic acid may encode polypeptides that may aid in purification and/orimmunogenicity and/or stability without shifting the codon reading frameof the transporter. In some embodiments, the fused nucleic acid willencode for a polypeptide to aid purification of the transporter. In someembodiments the fused nucleic acid will encode for an epitope and/or anaffinity tag. In other embodiments, the fused nucleic acid will encodefor a polypeptide that correlates to a site directed for, or prone to,cleavage. In preferred embodiments, the fused nucleic acid will encodefor polypeptides that are sites of enzymatic cleavage. In furtherembodiments, the enzymatic cleavage will aid in isolating thetransporter.

In other embodiments, the multiple nucleic acids will be fused to thenucleic acid encoding the transporters. The fused nucleic acids mayencode for polypeptides that aid purification and/or enzymatic cleavageand/or stability. In further embodiments, the fused nucleic acids willnot elongate the expressed polypeptide significantly.

In some embodiments the additional polypeptides may comprise an epitope.In other embodiments, the additional polypeptides may comprise anaffinity tag. By way of example, fusion of a polypeptide comprising anepitope and/or an affinity tag to a transporter may aid in purificationand/or identification of the polypeptide. By way of example, thepolypeptide segment may be a His-tag, a myc-tag, an S-peptide tag, a MBPtag (maltose binding protein), a GST tag (glutathione S-transferase), aFLAG tag, a thioredoxin tag, a GFP tag (green fluorescent protein), aBCCP (biotin carboxyl carrier protein), a calmodulin tag, a Strep tag,an HSV-epitope tag, a V5-epitope tag, and a CBP tag. The use of suchepitopes and affinity tags is known to those skilled in the art.

In further embodiments, the additional polypeptides may provide a fusionprotein comprising sites for cleavage of the polypeptide. The cleavagesites are useful for later cleaving the transporter from the fusedpolypeptides, such as with targeting polypeptides. As an example, apolypeptide may be cleaved by hydrolysis of the peptide bond. In someembodiments, the cleavage is performed by an enzyme. In some embodimentscleavage occurs in the cell. In other embodiments, cleavage occursthrough artificial manipulation and/or artificial introduction of acleaving enzyme. By way of example, cleavage enzymes may include pepsin,trypsin, chymotrypsin, and/or Factor Xa.

Fusion polypeptides may further possess additional structuralmodifications not shared with the same organically synthesized peptide,such as adenylation, carboxylation, glycosylation, hydroxylation,methylation, phosphorylation or myristylation. These added structuralmodifications may be further selected or preferred by the appropriatechoice of recombinant expression system. On the other hand, fusionpolypeptides may have their sequence extended by the principles andpractice of organic synthesis.

Generally, the fusion proteins of the invention may be producedaccording to techniques; which are described in the prior art. Forexample, these techniques involve recombinant techniques which can becarried out as described in Sambrook and Russell, Molecular Cloning: ALaboratory Manual, CSH Press, 2001 or in Volumes 1 and 2 of Ausubel,Current Protocols in Molecular Biology, Current Protocols, 1994.Accordingly, the individual portions of the fusion protein may beprovided in the form of nucleic acid molecules encoding them which arecombined and, subsequently, expressed in a host organism or in vitro.Alternatively, the provision of the fusion protein or parts thereof mayinvolve chemical synthesis or the isolation of such portions fromnaturally occurring sources, whereby the elements which may in part beproduced by recombinant techniques may be fused on the protein levelaccording to suitable methods, e.g. by chemical cross-linking forinstance as disclosed in WO 94/04686. Furthermore, if deemedappropriate, the fusion protein may be modified post-translationally inorder to improve its properties for the respective goal, e.g., toenhance solubility, to increase pH insensitivity, to be better toleratedin a host organism, to make it adherent to a certain substrate in vivoor in vitro, the latter potentially being useful for immobilizing thefusion protein to a solid phase etc. The person skilled in the art iswell aware of such modifications and their usefulness. Illustratingexamples include the modification of single amino acid side chains (e.g.by glycosylation, myristolation, phosphorylation, carbethoxylation oramidation), coupling with polymers such as polyethylene glycol,carbohydrates, etc. or with protein moieties, such as antibodies orparts thereof, or other enzymes etc.

In another embodiment of the invention, the fusion protein furthercomprises a targeting signal sequence. Transport of proteins to asubcellular compartment such as the chloroplast, vacuole, peroxisome,glyoxysome, cell wall or mitochondrion or for secretion into theapoplast, is accomplished by means of operably linking the nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of a geneencoding the protein of interest. Targeting sequences at the 5′ and/or3′ end of the structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lemer et al., Plant Physiol. 91:124-129(1989); Frontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal. Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

The term “targeting signal sequence” refers to amino acid sequences, thepresence of which in an expressed protein targets it to a specificsubcellular localization. For example, corresponding targeting signalsmay lead to the secretion of the expressed transporter, e.g. from abacterial host in order to simplify its purification. Preferably,targeting of the transporter may be used to affect the concentration ofa sugar in a specific subcellular or extracellular compartment.Appropriate targeting signal sequences useful for different groups oforganisms are known to the person skilled in the art and may beretrieved from the literature or sequence data bases.

The transporters of the present invention may be expressed in anylocation in the cell, including the cytoplasm, cell surface orsubcellular organelles such as the nucleus, vesicles, ER, vacuole, etc.Methods and vector components for targeting the expression of proteinsto different cellular compartments are well known in the art, with thechoice dependent on the particular cell or organism in which thebiosensor is expressed. See, for instance, Okumoto et al. PNAS 102:8740-8745, 2005; Fehr et al. J Fluoresc 14: 603-609, 2005, which areherein incorporated by reference in their entireties. Transport ofprotein to a subcellular compartment such as the chloroplast, vacuole,peroxisome, glyoxysome, cell wall or mitochondrion or for secretion intothe apoplast, may be accomplished by means of operably linking anucleotide sequence encoding a signal sequence to the 5′ and/or 3′region of a gene encoding the transporter. Targeting sequences at the 5′and/or 3′ end of the structural gene may determine during proteinsynthesis and processing where the encoded protein is ultimatelycompartmentalized.

If targeting to the plastids of plant cells is desired, the followingtargeting signal peptides can for instance be used: amino acid residues1 to 124 of Arabidopsis thaliana plastidial RNA polymerase (AtRpoT 3)(Plant Journal 17: 557-561, 1999); the targeting signal peptide of theplastidic Ferredoxin: NADP+ oxidoreductase (FNR) of spinach (Jansen etal., Current Genetics 13: 517-522, 1988) in particular, the amino acidsequence encoded by the nucleotides −171 to 165 of the cDNA sequencedisclosed therein; the transit peptide of the waxy protein of maizeincluding or without the first 34 amino acid residues of the mature waxyprotein (Klosgen et al., Mol. Gen. Genet. 217: 155-161, 1989); thesignal peptides of the ribulose bisphosphate carboxylase small subunit(Wolter et al., PNAS 85: 846-850, 1988; Nawrath et al., PNAS 91:12760-12764, 1994), of the NADP malat dehydrogenase (Gallardo et al.,Planta 197: 324-332, 1995), of the glutathione reductase (Creissen etal., Plant J. 8: 167-175, 1995) or of the R1 protein (Lorberth et al.,Nature Biotechnology 16: 473-477, 1998).

Targeting to the mitochondria of plant cells may be accomplished byusing the following targeting signal peptides: amino acid residues 1 to131 of Arabidopsis thaliana mitochondrial RNA polymerase (AtRpoT 1)(Plant Journal 17: 557-561, 1999) or the transit peptide described byBraun (EMBO J. 11: 3219-3227, 1992).

Targeting to the vacuole in plant cells may be achieved by using thefollowing targeting signal peptides: The N-terminal sequence (146 aminoacids) of the patatin protein (Sonnewald et al., Plant J. 1: 95-106,1991) or the signal sequences described by Matsuoka and Neuhaus (Journalof Exp. Botany 50: 165-174, 1999); Chrispeels and Raikhel (Cell 68:613-616, 1992); Matsuoka and Nakamura (PNAS 88: 834-838, 1991); Bednarekand Raikhel (Plant Cell 3: 1195-1206, 1991) or Nakamura and Matsuoka(Plant Phys. 101: 1-5, 1993).

Targeting to the ER in plant cells may be achieved by using, e.g., theER targeting peptide HKTMLPLPLIPSLLLSLSSAEF (SEQ ID NO: 118) inconjunction with the C-terminal extension HDEL (Haselhoff, PNAS 94:2122-2127, 1997). Targeting to the nucleus of plant cells may beachieved by using, e.g., the nuclear localization signal (NLS) of thetobacco C2 polypeptide QPSLKRMKIQPSSQP (SEQ ID NO: 119).

Targeting to the extracellular space may be achieved by using e.g. oneof the following transit peptides: the signal sequence of the proteinaseinhibitor II-gene (Keil et al., Nucleic Acid Res. 14: 5641-5650, 1986;von Schaewen et al., EMBO J. 9: 30-33, 1990), of the levansucrase genefrom Erwinia amylovora (Geier and Geider, Phys. Mol. Plant Pathol. 42:387-404, 1993), of a fragment of the patatin gene B33 from Solanumtuberosum, which encodes the first 33 amino acids (Rosahl et al., MolGen. Genet. 203: 214-220, 1986) or of the one described by Oshima et al.(Nucleic Acids Res. 18: 181, 1990).

Furthermore, targeting to the membrane may be achieved by using theN-terminal signal anchor of the rabbit sucrase-isomaltase (Hegner etal., J. Biol. Chem. 276: 16928-16933, 1992).

Targeting to the membrane in mammalian cells can be accomplished byusing the N-terminal myristate attachment sequence MGSSKSK (SEQ ID NO:120) or C-terminal prenylation sequence CaaX, where “a” is an aliphaticamino acid (i.e. Val, Leu or Ile) and “X” is any amino acid (Garabet,Methods Enzymol. 332: 77-87, 2001).

Additional targeting to the plasma membrane of plant cells may beachieved by fusion to a transporter, preferentially to the sucrosetransporter SUT1 (Riesmeier, EMBO J. 11: 4705-4713, 1992). Targeting todifferent intracellular membranes may be achieved by fusion to membraneproteins present in the specific compartments such as vacuolar waterchannels (γTIP) (Karlsson, Plant J. 21: 83-90, 2000), MCF proteins inmitochondria (Kuan, Crit. Rev. Biochem. Mol. Biol. 28: 209-233, 1993),triosephosphate translocator in inner envelopes of plastids (Flugge,EMBO J. 8: 39-46, 1989) and photosystems in thylacoids.

Targeting to the golgi apparatus can be accomplished using theC-terminal recognition sequence K(X)KXX (SEQ ID NO: 121) where “X” isany amino acid (Garabet, Methods Enzymol. 332: 77-87, 2001).

Targeting to the peroxisomes can be done using the peroxisomal targetingsequence PTS I or PTS II (Garabet, Methods Enzymol. 332: 77-87, 2001).

Targeting to the nucleus in mammalian cells can be achieved using theSV-40 large T-antigen nuclear localisation sequence PKKKRKV (SEQ ID NO:122) (Garabet, Methods Enzymol. 332: 77-87, 2001).

Targeting to the mitochondria in mammalian cells can be accomplishedusing the N-terminal targeting sequence MSVLTPLLLRGLTGSARRLPVPRAKISL(SEQ ID NO: 123) (Garabet, Methods Enzymol. 332: 77-87, 2001).

In some embodiments, expression of the transporter may be targeted toparticular tissue(s) or cell type(s). For example, a particular promotermay be used to drive transcription of a nucleic acid encoding thetransporter. A promoter is an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. A promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. A constitutive promoter is a promoter thatis active under most environmental and developmental conditions. Aninducible promoter is a promoter that is active under environmental ordevelopmental regulation. Any inducible promoter can be used, see, e.g.,Ward et al., Plant Mol. Biol. 22:361-366, 1993. Exemplary induciblepromoters include, but are not limited to, that from the ACEI system(responsive to copper) (Meft et al., Proc. Natl. Acad. Sci. USA90:4567-4571, 1993; 1n2 gene from maize (responsive tobenzenesulfonamide herbicide safeners) (Hershey et al., Mol. Gen.Genetics 227:229-237, 1991, and Gatz et al., Mol. Gen. Genetics243:32-38, 1994) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genetics 227:229-237, 1991). The inducible promoter may respond to anagent foreign to the host cell, see, e.g., Schena et al., PNAS 88:10421-10425, 1991.

The promoter may be a constitutive promoter. A constitutive promoter isoperably linked to a gene for expression or is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression. Many different constitutive promoters can beutilized in the instant invention. For example, in a plant cell,constitutive promoters include, but are not limited to, the promotersfrom plant viruses such as the 35S promoter from CaMV (Odell et al.,Nature 313: 810-812, 1985) and the promoters from such genes as riceactin (McElroy et al., Plant Cell 2: 163-171, 1990); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632, 1989, and Christensenet al., Plant Mol. Biol. 18: 675-689, 1992); pEMU (Last et al., Theor.Appl. Genet. 81:581-588, 1991); MAS (Velten et al., EMBO J. 3:2723-2730,1984) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics 231:276-285, 1992 and Atanassova et al., Plant Journal 2(3): 291-300, 1992).Prokaryotic promoter elements include those which carry optimal −35 and−10 (Pribnow box) sequences for transcription by RNA polymerase inEscherichia coli. Some prokaryotic promoter elements may containoverlapping binding sites for regulatory repressors (e.g. the Lac, andTAC promoters, which contain overlapping binding sites for lac repressorthereby conferring inducibility by the substrate homolog IPTG). Examplesof prokaryotic genes from which suitable promoter sequences may beobtained include E. coli lac, ara, and trp. Prokaryotic viral promoterelements of the present invention include lambda phage promoters (e.g.P_(RM) and P_(R)), T7 phage promoter elements, and SP6 promoterelements. Eukaryotic promoter vector elements of the invention includeboth yeast (e.g. GAL1, GAL10, CYC1) and mammalian (e.g. promoters ofglobin genes and interferon genes). Further eukaryotic promoter vectorelements include viral gene promoters such as those of the SV40promoter, the CMV promoter, herpes simplex thymidine kinase promoter, aswell as any of various retroviral LTR promoter elements (e.g. the MMTVLTR (SEQ ID NO: 124)). Other eukaryote examples include the the hMTIIapromoters (e.g. U.S. Pat. No. 5,457,034), the HSV-1 4/5 promoter (e.g.U.S. Pat. No. 5,501,979), and the early intermediate HCMV promoter (WO92/17581).

The promoter may be a tissue-specific or tissue-preferred promoters. Atissue specific promoter assists to produce the transporter exclusively,or preferentially, in a specific tissue. Any tissue-specific ortissue-preferred promoter can be utilized. In plant cells, for examplebut not by way of limitation, tissue-specific or tissue-preferredpromoters include, a root-preferred promoter such as that from thephaseolin gene (Murai et al., Science 23: 476-482, 1983, andSengupta-Gopalan et al., PNAS 82: 3320-3324, 1985); a leaf-specific andlight-induced promoter such as that from cab or rubisco (Simpson et al.,EMBO J. 4(11): 2723-2729, 1985, and Timko et al., Nature 318: 579-582,1985); an anther-specific promoter such as that from LAT52 (Twell etal., Mol. Gen. Genetics 217: 240-245, 1989); a pollen-specific promotersuch as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168, 1993) or a microspore-preferred promoter such as that from apg(Twell et al., Sex. Plant Reprod. 6: 217-224, 1993).

Furthermore, the present invention relates to expression cassettescomprising the above-described nucleic acid molecule of the inventionand operably linked to control sequences allowing expression inprokaryotic or eukaryotic cells.

In a further embodiment, the invention relates to a method for producingcells capable of expressing the transporters of the invention comprisinggenetically engineering cells with an above-described nucleic acidmolecule, expression cassette or vector of the invention.

Another embodiment of the invention relates to host cells, in particularprokaryotic or eukaryotic cells, genetically engineered with anabove-described nucleic acid molecule, expression cassette or vector ofthe invention, and to cells descended from such transformed cells andcontaining a nucleic acid molecule, expression cassette or vector of theinvention and to cells obtainable by the above-mentioned method forproducing the same.

The host cells may be bacterial, fungal, insect, plant or animal hostcells. In one embodiment, the host cell is genetically engineered insuch a way that it contains the introduced nucleic acid molecule stablyintegrated into the genome. In another embodiment, the nucleic acidmolecule can be expressed so as to lead to the production of the fusionprotein of the invention.

An overview of different expression systems is for instance contained inMethods in Enzymology 153: 385-516, 1987, in Bitter et al. (Methods inEnzymology 153: 516-544, 1987) and in Sawers et al. (AppliedMicrobiology and Biotechnology 46: 1-9, 1996), Billman-Jacobe (CurrentOpinion in Biotechnology 7: 500-4, 1996), Hockney (Trends inBiotechnology 12: 456-463, 1994), and Griffiths et al., (Methods inMolecular Biology 75: 427-440, 1997). An overview of yeast expressionsystems is for instance given by Hensing et al. (Antoine von Leuwenhoek67: 261-279, 1995), Bussineau (Developments in BiologicalStandardization 83: 13-19, 1994), Gellissen et al. (Antoine vanLeuwenhoek 62: 79-93, 1992), Fleer (Current Opinion in Biotechnology 3:486-496, 1992), Vedvick (Current Opinion in Biotechnology 2: 742-745,1991) and Buckholz (Bio/Technology 9: 1067-1072, 1991).

Expression vectors have been widely described in the literature. As arule, they contain not only a selection marker gene and a replicationorigin ensuring replication in the host selected, but also a bacterialor viral promoter and, in most cases, a termination signal fortranscription. Between the promoter and the termination signal, there isin general at least one restriction site or a polylinker which enablesthe insertion of a coding nucleotide sequence. It is possible to usepromoters ensuring constitutive expression of the gene and induciblepromoters which permit a deliberate control of the expression of thegene. Bacterial and viral promoter sequences possessing these propertiesare described in detail in the literature. Regulatory sequences for theexpression in microorganisms (for instance E. coli, S. cerevisiae) aresufficiently described in the literature. Promoters permitting aparticularly high expression of a downstream sequence are for instancethe T7 promoter (Studier et al., Methods in Enzymology 185: 60-89,1990), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez andChamberlin (Eds), Promoters, Structure and Function; Praeger, N.Y.,1982, p. 462-481; DeBoer et al., PNAS 80: 21-25, 1983), Ip1, rac (Boroset al., Gene 42: 97-100, 1986). Inducible promoters may be used for thesynthesis of proteins. These promoters often lead to higher proteinyields than do constitutive promoters. In order to obtain an optimumamount of protein, a two-stage process is often used. First, the hostcells are cultured under optimum conditions up to a relatively high celldensity. In the second step, transcription is induced depending on thetype of promoter used. In this regard, a tac promoter is particularlysuitable which can be induced by lactose or IPTG(isopropyl-.beta.-D-thiogalactopyranoside) (DeBoer et al., PNAS 80:21-25, 1983). Termination signals for transcription such as theSV40-poly-A site or the tk-poly-A site useful for applications inmammalian cells are also described in the literature. Suitableexpression vectors are known in the art such as Okayama-Berg cDNAexpression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1 (GIBCO BRL)) or pCI (Promega).

The invention also includes host cells transfected with a vector or anexpression vector encoding the transporters of the invention, includingprokaryotic cells, such as E. coli or other bacteria, or eukaryoticcells, such as yeast cells or animal cells. The living cell cultures maycomprise prokaryotic cells or eukaryotic cells. Examples of sources forprokaryotic cells include but are not limited to bacteria or archaea.Examples of sources for eukaryotic cells include but are not limited to:yeast, fungi, protists, mammals, arthropods, humans, animals, molluscs,annelids, nematodes, crustaceans, platyhelminthes, monotremes, fish,marsupials, reptiles, amphibians, birds, rodents, insects, and plants.

The transformation of the host cell with a nucleic acid molecule orvector according to the invention can be carried out by standardmethods, as for instance described in Sambrook and Russell, MolecularCloning: A Laboratory Manual, CSH Press, 2001; Methods in YeastGenetics, A Laboratory Course Manual, Cold Spring Harbor LaboratoryPress, 1990). For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas, e.g., calcium phosphate orDEAE-Dextran mediated transfection or electroporation may be used forother cellular hosts. The host cell is cultured in nutrient mediameeting the requirements of the particular host cell used, in particularin respect of the pH value, temperature, salt concentration, aeration,antibiotics, vitamins, trace elements etc. The transporters according tothe present invention can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromography and lectinchromatography. A ligand or substrate, such as glucose, for thetransporter may by used for affinity purification or a fusion protein ofthe transporter may be purified by applying an affinity chromatographywith a substrate or ligand to which the fused portion binds, such as anaffinity tag. Protein refolding steps can be used, as necessary, incompleting the configuration of the protein. Finally, high performanceliquid chromatography (HPLC) can be employed for final purificationsteps.

Accordingly, a further embodiment of the invention relates to a methodfor producing the transporters of the invention comprising culturing theabove-described host cells under conditions allowing the expression ofsaid transporters and recovering said transporters from the culture.Depending on whether the expressed protein is localized in the hostcells or is secreted from the cell, the protein can be recovered fromthe cultured cells and/or from the supernatant of the medium.

Alternatively, the transporter may be delivered to the cell usingmicroinjection, particle bombardment, introduction of embedded sensors,or by fusion of a peptide sequence that leads to uptake of the biosensorinto cells.

Moreover, the invention relates to transporters which are obtainable bya method for their production as described above.

The transporters of the present invention may, e.g., be a product ofchemical synthetic procedures or produced by recombinant techniques froma prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect or mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the expressedtransporters may be glycosylated or may be non-glycosylated. Thetransporters of the invention may also include an initial methionineamino acid residue. The transporters according to the invention may befurther modified to contain additional chemical moieties not normallypart of the protein. Those derivatized moieties may, e.g., improve thestability, solubility, the biological half life or absorption of theprotein. The moieties may also reduce or eliminate any undesirable sideeffects of the protein and the like. An overview for these moieties canbe found, e.g., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa.).

Transgenics

The present invention provides transgenic plants and non-humantransgenic organisms, i.e. multicellular organisms, comprising a nucleicacid molecule encoding the transporters of the invention, such as GLUEs,or an expression cassette or vector as described above, stablyintegrated into its genome, at least in a subset of the cells of thatorganism, or to parts thereof such as tissues or organs.

The present invention provides transgenic plants or plant tissuecomprising transgenic plant cells, i.e. comprising stably integratedinto their genome, an above-described nucleic acid molecule, expressioncassette or vector of the invention. The present invention also providestransgenic plants, plant cells or plant tissue obtainable by a methodfor their production as outlined below.

In one embodiment, the present invention provides a method for producingtransgenic plants, plant tissue or plant cells comprising theintroduction of a nucleic acid molecule, expression cassette or vectorof the invention into a plant cell and, optionally, regenerating atransgenic plant or plant tissue therefrom. The transgenic plantsexpressing the transporter can be of use in affecting the transport ofsugars throughout and between the organs of an organism, such as to orfrom the soil. The transgenic plants expressing transporters of theinvention can be of use for investigating metabolic or transportprocesses of, e.g., organic compounds with a timely and spatialresolution that was not achievable in the prior art.

Methods for the introduction of foreign nucleic acid molecules intoplants are well-known in the art. For example, plant transformation maybe carried out using Agrobacterium-mediated gene transfer,microinjection, electroporation or biolistic methods as it is, e.g.,described in Potrykus and Spangenberg (Eds.), Gene Transfer to Plants.Springer Verlag, Berlin, N.Y., 1995. Therein, and in numerous otherprior art references, useful plant transformation vectors, selectionmethods for transformed cells and tissue as well as regenerationtechniques are described which are known to the person skilled in theart and may be applied for the purposes of the present invention.

In another aspect, the invention provides harvestable parts and methodsto propagation material of the transgenic plants according to theinvention which contain transgenic plant cells as described above.Harvestable parts can be in principle any useful part of a plant, forexample, leaves, stems, fruit, seeds, roots etc. Propagation materialincludes, for example, seeds, fruits, cuttings, seedlings, tubers,rootstocks etc.

In certain aspects, the invention provides a transgenic non-human animalhaving a phenotype characterized by expression of the nucleic acidsequence coding for the expression of the transporters. The phenotype isconferred by a transgene contained in the somatic and germ cells of theanimal, which may be produced by (a) introducing a transgene into azygote of an animal, the transgene comprising a DNA construct encodingthe transporters; (b) transplanting the zygote into a pseudopregnantanimal; (c) allowing the zygote to develop to term; and (d) identifyingat least one transgenic offspring containing the transgene. The step ofintroducing the transgene into the embryo may include introducing anembryonic stem cell containing the transgene into the embryo, orinfecting the embryo with a retrovirus containing the transgene.Preferred transgenic animals will express the encoded transporters.Transgenic animals of the invention include transgenic S. cerevisae, C.elegans, Drosophila, particularly, D. melanogaster, and transgenic miceand other animals.

The invention also provides a transgenic non-human animal comprising atleast one nucleic acid molecule encoding a transporter, expressioncassette or vector comprising the nuceliec acid which may be stablyintegrated into their genome.

The present invention also encompasses a method for the production of atransgenic non-human animal comprising introducing a nucleic acidmolecule, expression cassette or vector of the invention into a germcell, an embryonic cell, stem cell or an egg or a cell derivedtherefrom. It is preferred that such transgenic animals expressing thetransporter of the invention or any developmental stage thereof startingfrom the zygote may be used as model organisms where it is possible todetermine the distribution of a certain compound (depending on theenzyme present in the fusion protein) in real time without disruptingtissue integrity. These model organisms may be particularly useful fornutritional or pharmacological studies or drug screening. Production oftransgenic embryos and screening of them can be performed, e.g., asdescribed by A. L. Joyner (Ed.), Gene Targeting, A Practical Approach,Oxford University Press, 1993. The DNA of the embryos can be analyzedusing, e.g., Southern blots with an appropriate probe or based on PCRtechniques.

A transgenic non-human animal in accordance with the invention may,e.g., be a transgenic mouse, rat, hamster, marsupial, monotreme, dog,monkey, rabbit, chiroptera, pig, frog, nematode such as Caenorhabditiselegans, fruitfly such as Drosophila melanogaster, or fish suchtorpediniforms, such as torpedo fish, tetraodontiforms, characiforms,lamniforms, or cypriniforms, such as zebrafish, comprising a nucleicacid molecule, expression cassette or vector of the invention,preferably stably integrated into its genome, or obtained by the methodmentioned above. Such a transgenic non-human animal may comprise one orseveral copies of the same or different nucleic acid molecules of theinvention. The presence of a nucleic acid molecule, expression cassetteor vector of the invention in such a transgenic non-human animal leadsto the expression of the transporter of the invention. The transgenicnon-human animal of the invention has numerous utilities, including as aresearch model. Accordingly, in this instance, the mammal is preferablya laboratory animal such as a chimpanzee, mouse, or rat.

Thus, in one embodiment, the transgenic non-human animal of theinvention is a mouse, a rat, a dog, such as a beagle, or a zebrafish.Numerous reports revealed that said animals are particularly well suitedas model organisms for the investigation of the drug metabolism and itsdeficiencies or cancer. Advantageously, transgenic animals can be easilycreated using said model organisms, due to the availability of varioussuitable techniques well known in the art for investigating sugartransport, particularly glucose transport.

Antibodies

Another aspect of the invention is directed to the generation ofantibodies that bind to the transporters of the invention. Examples ofantibodies encompassed by the present invention, include, but are notlimited to, antibodies specific for the transporters of the claimedinvention and neutralizing antibodies. The antibodies of the inventionmay be characterized using methods well known in the art.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Antibodiesmay be derived from murine, rat, human, primate, or any other origin(including chimeric and humanized antibodies).

In one embodiment, the antibodies may be polyclonal or monoclonalantibodies. Methods of preparing monoclonal and polyclonal antibodiesare well known in the art.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts and includes antibody fragments as defined herein.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al. (1975) Nature256, 495 or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222, 581-597,for example. “Polyclonal” antibodies refer to a selection of antibodiesdirected against a particular protein or fragment thereof, wherein theantibodies may bind to different epitopes.

In other embodiments, the antibodies may be humanized by methods knownin the art. A humanized antibody is an immunoglobulin molecule thatcontains minimal sequence derived from non-human immunoglobulin. In yetother embodiments, fully human antibodies are obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. In other embodiments, theantibodies are chimeric. A chimeric antibody is an antibody thatcombines characteristics from two different antibodies. Methods ofpreparing chimeric antibodies are known in the art.

In other embodiments, the nucleotide sequence that encodes the antibodyis obtained and then cloned into a vector for expression or propagation.In another embodiment, antibodies are made recombinantly and expressedusing methods known in the art. By way of example, transporters orfragments thereof may be used as an antigen for the purposes ofisolating recombinant antibodies by these techniques. Antigenic motifsof the transporters can readily be determined by methods known in theart, such as for example the Jameson-Wolf method (CABIOS, 4: 181-186,1988). Antibodies can be made recombinantly by using the gene sequenceto express the antibody recombinantly in host cells. Methods for makingderivatives of antibodies and recombinant antibodies are known in theart.

In other embodiments, the antibodies are bound to a carrier byconventional methods in the art, for use in, for example, isolating orpurifying native transporters or detecting native transporters in abiological sample or specimen.

The term “antibodies or fragments thereof” as used herein refers toantibodies or fragments thereof that specifically bind to a sugartransporter or a fragment thereof and do not specifically bind to othernon-transporters. Antibodies or fragments that immunospecifically bindto a transporter or fragment thereof do not non-specifically cross-reactwith other antigens (e.g., binding cannot be competed away with anon-transporter, e.g., BSA in an appropriate immunoassay). Antibodies orfragments that immunospecifically bind to a transporter can beidentified, for example, by immunoassays or other techniques known tothose of skill in the art. Antibodies of the invention include, but arenot limited to, synthetic antibodies, monoclonal antibodies, heavy-chainonly antibodies, recombinantly produced antibodies, intrabodies,diabodies, multispecific antibodies (including bi-specific: antibodies),human antibodies, humanized antibodies, chimeric antibodies,single-chain Fvs (scFv) (including bi-specific scfvs), single chainantibodies, Fab′ fragments, F(ab′)2 fragments, disulfide-linked Fvs(sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above. In particular, antibodies of the presentinvention include immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site that immunospecifically binds to a transporter(e.g., one or more complementarity determining regions (CDRs) of ananti-transporter antibody).

As used herein, an “intact” antibody is one which comprises anantigen-binding site as well as a C_(L) and at least heavy chainconstant domains, C_(H1) and C_(H2) and C_(H3). The constant domains maybe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variant thereof. Preferably,the intact antibody has one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding CDR or variable region of the intactantibody. Examples of antibody fragments include Fab, Fv, Fab′ andF(ab′)₂ fragments; diabodies; linear antibodies (see U.S. Pat. No.5,641,870 and Zapata et al. (1995) Protein Eng. 8, 1057-1062);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H1)). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H1) domain including oneor more cysteines from the antibody hinge region. Fab′-S_(H) is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

As used herein, “Fv” is the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragment consistsof a dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanate six hypervariable loops (three loops each from the H and Lchain) that contribute the amino acid residues for antigen binding andconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

As used herein, “Single-chain Fv” also abbreviated as “sFv” or “scFv”are antibody fragments that comprise the VH and VL antibody domainsconnected into a single polypeptide chain. The scFv polypeptide mayfurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the sFv to form the desired structure for antigenbinding (see Rosenburg et al. (1994) The Pharmacology of MonoclonalAntibodies, Springer-Verlag, pp. 269-315).

As used herein, the term “diabodies” refers to small antibody fragmentsprepared by constructing sFv fragments (see preceding paragraph) withshort linkers (about 5 to about 10 residues) between the V_(H) and V_(L)domains such that inter-chain but not intra-chain pairing of the Vdomains is achieved, resulting in a bivalent fragment, i.e., fragmenthaving two antigen-binding sites. Bispecific diabodies are heterodimersof two “crossover” sFv fragments in which the V_(H) and V_(L) domains ofthe two antibodies are present on different polypeptide chains.Diabodies are described more fully in, for example, WO 93/11161 andHollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6444-6448.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous components. In preferred embodiments, the antibodywill be purified to greater than 95% by weight of antibody, and mostpreferably more than 99% by weight. Isolated antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

In one embodiment of the invention, the conjugated antibody binds to anepitope on the cytoplasmic domain of a protein specific to cancer cells(i.e., a cancer cell marker). In another embodiment, the conjugatedantibody includes, but is not limited to, an antibody which binds to anepitope on the cytoplasmic domain of sF.

Pharmaceutical Compositions

Another aspect of the invention is directed toward the use of thetransporters as part of a pharmaceutical composition. The antibodies andnucleic acids of the present invention may also be used as part of apharmaceutical composition. The compositions used in the methods of theinvention generally comprise, by way of example and not limitation, andeffective amount of a nucleic acid or polypeptide (e.g., an amountsufficient to induce an immune response) of the invention or antibody ofthe invention (e.g., an amount of a neutralizing antibody sufficient tomitigate infection, alleviate a symptom of infection and/or preventinfection). The nucleic acids, polypeptides, and antibodies of theinvention can further comprise pharmaceutically acceptable carriers,excipients, or stabilizers known in the art (see generally Remington,(2005) The Science and Practice of Pharmacy, Lippincott, Williams andWilkins).

The nucleic acids, polypeptides, and antibodies of the present inventionmay be in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers may be nontoxic torecipients at the dosages and concentrations that are administered.Carriers, excipients or stabilizers may further comprise buffers.Examples of buffers include, but are not limited to, carbohydrates (suchas monosaccharide and disaccharide), sugars (such as sucrose, mannitol,and sorbitol), phosphate, citrate, antioxidants (such as ascorbic acidand methionine), preservatives (such as phenol, butanol, benzanol; alkylparabens, catechol, octadecyldimethylbenzyl ammonium chloride,hexamethoniuni chloride, resorcinol, cyclohexanol, 3-pentanol,benzalkonium chloride, benzethonium chloride, and m-cresol), lowmolecular weight polypeptides, proteins (such as serum albumin orimmunoglobulins), hydrophilic polymers amino acids, chelating agents(such as EDTA), salt-forming counter-ions, metal complexes (such asZn-protein complexes), and non-ionic surfactants (such as TWEEN™ andpolyethylene glycol).

The pharmaceutical composition of the present invention can furthercomprise additional agents that serve to enhance and/or complement thedesired effect. By way of example, to enhance the immunogenicity of atransporter of the invention, the pharmaceutical composition may furthercomprise an adjuvant. Adjuvants include aluminum salts (alum), CompleteFreund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Muramyldipeptide (MDP), synthetic analogues of MDP,N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide(MTP-PE) and compositions containing a metabolizable oil and anemulsifying agent, wherein the oil and emulsifying agent are present inthe form of an oil-in-water emulsion having oil droplets substantiallyall of which are less than one micron in diameter (see, for example, EP0399843).

In some embodiments, the adjuvant comprises a Toll like receptor (TLR) 4ligand, in combination with a saponin. The Toll like receptor (TLR) 4ligand may be for example, an agonist such as a lipid A derivativeparticularly monophosphoryl lipid A or more particularly 3 Deacylatedmonophoshoryl lipid A (3 D-MPL). 3 D-MPL is sold under the trademarkMPL® by Corixa Corporation and primarily promotes CD4+ T cell responseswith an IFN-g (Th1) phenotype. It can be produced according to themethods disclosed in GB 2220211A. Chemically, it is a mixture of3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains.In one embodiment in the compositions of the present invention smallparticle 3 D-MPL is used. Small particle 3 D-MPL has a particle sizesuch that it may be sterile-filtered through a 0.22 μm filter. Suchpreparations are described in PCT Patent Application WO 94/21292.

The adjuvant may also comprise one or more synthetic derivatives oflipid A which are known to be TLR 4 agonists including, but not limitedto: OM174(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyldihydrogenphosphate),as described in PCT Patent Application WO 95/14026; OM 294 DP(3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate),as described in WO 9964301 and WO 00/0462; and, OM 197 MP-Ac DP(3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate10-(6-aminohexanoate) (WO 01/46127).

Other TLR4 ligands which may be used include, but are not limited to,alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO98/50399 or U.S. Pat. No. 6,303,347 (processes for preparation of AGPsare also disclosed), or pharmaceutically acceptable salts of AGPs asdisclosed in U.S. Pat. No. 6,764,840. Some AGPs are TLR4 agonists, andsome are TLR4 antagonists. Both can be used as one or more adjuvants inthe compositions of the invention.

A saponin carrier for use in the present invention is Quil A and itsderivatives. Quil A is a saponin preparation isolated from the SouthAmerican tree Quilaja Saponaria Molina and was first described as havingadjuvant activity by Dalsgaard et al. (1974) Saponin adjuvants, Archiv.für die gesamte Virusforschung, Vol. 44, Springer Verlag, pp. 243-254.Purified fragments of Quil A have been isolated by HPLC which retainadjuvant activity without the toxicity associated with Quil A (EP 0 362278), for example QS7 and QS21 (also known as QA7 and QA21). QS21 is anatural saponin derived from the bark of Quillaja saponaria Molina whichinduces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2aantibody response and is a preferred saponin in the context of thepresent invention.

Particular formulations of QS21 have been described which areparticularly preferred, these formulations further comprise a sterol (WO96/33739). The saponins forming part of the present invention may beseparate in the form of micelles, mixed micelles (preferentially, butnot exclusively with bile salts) or may be in the form of ISCOM matrices(EP 0109942 B1), liposomes or related colloidal structures such asworm-like or ring-like multimeric complexes or lipidic/layeredstructures and lamellae when formulated with cholesterol and lipid, orin the form of an oil in water emulsion (for example as in WO 95/17210).The saponins may be associated with a metallic salt, such as aluminiumhydroxide or aluminium phosphate (WO 98/15287). In some embodiments, thesaponin is presented in the form of a liposome, ISCOM or an oil in wateremulsion.

In some embodiments, adjuvants are combinations of 3D-MPL and QS21 (EP0671948 B1) and oil in water emulsions comprising 3D-MPL and QS21 (WO95/17210, WO 98/56414).

Methods of Using Sugar Transporters

The present invention provides for methods of using the sugartransporters, for example GLUE. The methods comprise introducing thesugar transporters into a cell, such as a cell in vitro or the cell ofan organism. The sugar transporter introduced into a cell may be awild-type or mutant transporter. The transporter may be introduced as anucleic acid encoding the transporter or as an amino acid polypeptide.The ability of the transporter to function will be apparent to thoseskilled in the art based on the desired outcome. For example, aconstitutively active or a wild-type transporter may be used to overcomea sugar transport deficiency. A mutant transporter may be used toovercome a problem with sugar transport. A variant of a sugar transportmay be introduced to alter the desired gene expression of a pathogen.

The methods of the present invention provide for altering thedevelopment of an organism. The organism may be an adult or an embryo.Introduction into a cell of the organism may affect the development ofthe organism. In a plant, the introduction of the sugar transporters mayaffect leaf development, shoot development, nectar development, rootdevelopment, anther development, xylem development, reproductivedevelopment, stem development, and fruit development. In an animal, theintroduction of the sugar transporters of the claimed invention mayaffect development of an organs, such as the brain heart, lungs,circulatory system, skin, liver, kidney, brain, spine, bones, muscle(smooth and skeletal), limbs, lugs, spleen, intestines, pancreas,adrenal glands, gall bladder, testes, ovaries, prostate, bladder,stomach, thyroid, parathyroid, hypothalamus, hippocampus, pineal gland,lymph nodes, mammary glands, immune system, or ductal systems.

The methods of the present invention may provide for affecting thefunctioning systems between organs of an organism, such as thecirculatory systems, nervous system (both sympathetic andparasympathetic) respiratory system, digestive system, excretory system,and reproductive system. In plants, introduction of the sugartransporters may affect sugar transport between the root and the stemand the leaves. In an animal, introduction of the sugar transporters mayaffect milk production, development and functioning of the reproductiveglands, ovulation, oxygen and carbon dioxide exchange, digestion of foodand adsorption of nutrients.

The methods of the present invention provide novel mechanisms foraffecting the susceptibility of attack to an organism, such as apathogen attack. A pathogen may be a prokaryote or eukaryote. A pathogenmay be a bacterium, a virus, a fungus, a worm, or an insect. Thepathogen may affect gene regulation of a host organism to providenutrients or sustinance to the pathogen, such as through hostsusceptible genes. The transporters of the present invention may alterthe pathogens ability to affect the host organisms gene transcription.The sugar transporters of the claimed invention introduced into the hostcell may defend the host cell from pathogen attack.

Those skilled in the art will appreciate that similar efflux steps arerequired to supply developing pollen, germinating pollen, developingembryos and all other cases where cells are exchanging carbon through anapoplasmic route (cell to cell via cell wall). A GLUE homolog, RPG1, islocalized in the tapetum and a mutation in RPG1 leads to inviability ofpollen (Guan Y F, Huang X Y, Zhu J, Gao J F, Zhang H X Yang Z N. 2008Plant Physiol. 147:852-63). Thus manipulation of the transporters mayaffect allocation.

The transporters of the present invention may modulate the secretion ofsugars, such as glucose, into the rhizosphere of a plant. For example,plants secrete 1.5 t/ha carbon into soil per vegetation period. Themethods of the present invention may allow for manipulation of sugarsecretion which in turn may affect plant productivity. By way ofexample, an increase in sugar secretion may attract more microorganismsto the cell, and may deposit more carbon into the soil (e.g. sequesteratmospheric CO₂). Reduction in sugar secretion may lead to increasedbiomass in plant. The general concept of such transporters in roots hasbeen described in part in Chaudhuri et al. 2008, Plant Journal.

The transporters of the present invention may modulate the secretion ofsugars, such as glucose, into the phyllosphere of a plant. For example,modulation in sugar secretion in the phyllopsphere may attract morebeneficial microorganisms or feed pathogens. Manipulation (also throughdevelopment of specific chemical inhibitors as pesticides)

The transporters of the present invention may modulate the secretion ofsugars, such as glucose, to affect the pollination patterns of a plant.The manipulation of sugar secretion may affect pollination patterns. Forexample, altered sugar secretion may attract different pollinators asdifferent pollinators require different nectar composition (Ge et al.Plant J. 24:725-734, 2000).

The transporters of the present invention may modulate the secretion ofsugars, such as glucose, and affect the development of the leaf andphloem of a plant. For example, Ge et al. have demonstrated thatoverexpression of a related protein leads to stimulation of phloemdevelopment and alters the symmetry of the leaf. Plant architecture mayalso be manipulated by the methods described herein.

Another aspect of the invention concerns methods to modulate pathogenactivity towards a plant and the cells of the plant. Pathogens(including symbionts) recruit certain transporters to feed them (Yang etal. 2006 PNAS 103:10503-10508). Yang et al. consider the gene asusceptibility factor, which is induced by a type IIIsecretion-system-dependent mechanism in a pathovar-specific way. Byanalyzing microarray data it can demonstrated that different pathogensrecruit different members of the GLUE family. One means by whichpathogens may recruit transporters is to affect the promoter region ofthe nucleic acid encoding the transporter to increase the number oftransporters present in a cell or their activity. Manipulation, such asintroducing different transporters or introducing different promotersupstream of the transporter may prevent pathogen infections and improveor transfer symbionts. Chemical inhibitors may be identified that blockthe transporter and thus prevent pathogen infection. Export of sugarsfrom leaves requires not only a proton sucrose cotransporter for phloemloading, but also cellular effluxers for export from mesophyll or phloemcells. Manipulation can affect plant productivity.

Milk is an important nutrient source for newborns, children and adults.In the US, milk consumption exceeds 80 liters per capita(www.foodsci.uoguelph.ca/dairyedu/intro.html). Milk is also used toproduce butter, yoghurt and cheese. In mammals, milk represents theprimary source of nutrition for newborns. Mother's milk and cow milkprovide many important nutrients as well as antibodies to the newborns.Besides proteins and lipids, milk contains also soluble sugars such asglucose and the disaccharide lactose. The lactose content of human milkis ˜7% (200 mM), that of bovine milk ˜4.5% (140 mM). Lactose is producedin alveolar cells that line the milk ducts of mammary glands.Specifically, lactose synthesis occurs in the Golgi, mediated by theheteromeric enzyme lactose synthase consisting of anβ-1,4-galactosyltransferase subunit and lactalbumin, which is highlyinduced during lactation.

The precursor glucose is imported through the basal membrane into theglandular cells by glucose transporters belonging to the GLUT and SGLTfamilies. Both glucose and UDP-galactose transporters are required atthe Golgi for import of the precursors for lactose synthesis, howeverthe Golgi glucose importer has not been identified. Lactose is assumedto occur by exocytosis (2). Since the membrane of the Golgi vesiclesappears to be impermeable to disaccharides, the high osmotic potentialattracts water import. During exocytosis, the water will be exported,contributing a major fraction of the water content of the milk.Understanding the cellular machinery contributing to lactose synthesisis thus important for multiple aspects of milk production andcomposition. Moreover, lactose content in bovine milk represents healthissues for large parts of the population due to the inability toefficiently metabolize lactose by lactase in the intestine. Lactasedeficiencies can be congenital (rare mutations affecting lactaseactivity in the intestine), or acquired (secondary lactase deficiency).The most common cause of lactase deficiency is a decrease in the amountof lactase that occurs after childhood and persists into adulthood,referred to as adult-type hypolactasia. Almost 100% of the Asianpopulation suffers from hypolactasia, leading to the necessity to eatlactose-free diets. Thus the present invention may be utilized to alterthe production of milk in a subject. The present invention may alter acell's ability to import or export lactase.

EXAMPLES

Signaling cascades that control nutrient uptake and metabolism as wellas the exchange of nutrients in biotic interactions with plants, e.g.nectar production in flowers to attract pollinators, the secretion ofsugars by the plant root into the rhizosphere to feed microorganisms andthe hijacking of these systems by pathogens. A novel transporterinvolved in supplying reproductive cells, nectaries, the rhizosphere andpathogens with sugars was recently identified. It was then found thatthis plant transporter has homologs in animals, specifically in mammalswhere the protein show many of the features of being involved in sugarsecretion in mammary glands. FRET nanosensors provide a unique toolenabling quantitative flux analysis with subcellular resolution (Okumotoet al. New Phytol. 180:271-295, 2008). These nanosensors are composed ofbacterial periplasmic binding proteins serving as recognition elements,coupled allosterically to a pair of spectral variants of the GreenFluorescent Protein (GFP) as reporter elements (Fehr et al. Proc. Natl.Acad. Sci. USA 99:9846-9851, 2002;Fehr et al. J. Biol. Chem.278:19127-19133, 2003; Deuschle et al. Protein Sci 14:2304-2314, 2005).

Conformational changes induced by ligand-binding to the recognitionelement translate into a change in fluorescence resonance energytransfer (FRET) between attached cyan and yellow fluorescent proteinmoieties. These sensors can be introduced genetically into living cells,permitting non-invasive measurements of analyte levels in living cellsand tissues (Fehr et al. J. Biol. Chem. 278:19127-19133, 2003). Throughthese sensors, glucose flux in intact Arabidopsis roots (Chaudhuri etal. Plant J. 56:948-962, 2008), glutamate release from hippocampalneurons (Okumoto et al. Proc Natl Acad Sci USA 102:8740-8745, 2005), andtryptophan exchange in cancer cells (Kaper et al. PLoS Biol 5:e257,2007) has been determined.

Example 1 Analysis of Glucose Flux Across the ER Membrane

To determine analyte levels inside organelles, these FRET nanosensorswere targeted to the respective subcellular compartments (Fehr et al. J.Fluoresc. 14:603-609, 2004). In order to directly monitor glucose fluxacross the ER membrane, FRET glucose sensors were targeted to the ERlumen by flanking them with an ER signal sequence and a KDEL retentionsignal. This approach permitted identification of high glucose fluxrates across the ER membrane, and suggested the existence of rapidbidirectional high-capacity transport activities for glucose in HepG2cells (Fehr et al. Mol Cell Biol 25:11102-11112, 2005).

Example 2 Identification of a Novel Sugar Transport Function in PlantRoots

Although soil contains only traces of soluble carbohydrates, plant rootsefficiently take up glucose and sucrose when supplied in artificialmedia. Soluble carbohydrates and other small metabolites found in soilare in part derived from exudation from plant roots. The molecularnature of the transporters for uptake and exudation is unknown. FRETglucose and sucrose sensors were deployed to characterize accumulationand elimination of glucose and sucrose in Arabidopsis roots tips(Chaudhuri et al. Plant J. 56:948-962, 2008). Glucose and sucroseaccumulation was insensitive to protonophores, and was similar at pH5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediatedby a novel class of proton-independent transport systems. Moreover, asopposed to all known plant glucose transporters, this new root transportsystem did not mediate transport of the glucose analog3-O-methylglucose.

Example 3 HEK293T Cells as an Expression System for Glucose Transporters

To be able to characterize glucose transport across the plasma membraneas well as the ER membrane better, cell lines with low endogenous sugaruptake capacity were assayed. It was found that HEK293T cells had verylow endogenous uptake and can be used as an expression system to definethe properties of GLUT and SGLT sugar transporters with the help of FRETglucose sensors.

Example 4 Identification of a Glucose Transporter Involved in NectarProduction and Rhizosphere Secretion

By utilizing the above described HEK293T cell system, a novel class ofArabidopsis sugar transporters was identified that are involved innectar production in plants and with all hallmarks of the glucosetransport activity described in root tips. A screening assay was theninitiated in which candidate transporter genes from Arabidopsis werecoexpressed with the FRET glucose sensor in the HEK293T cell expressionsystem. It was found that a member of an unknown class of membraneproteins (named GLUE1) induced glucose concentration-dependent FRETresponses (see, e.g., FIGS. 3 and 4).

It was then verified that none of the known mammalian GLUT or SGLTtransporter genes was induced when GLUE1 was expressed in the HEK293Tcells, and showed that GLUE1-mediated uptake was insensitive to the GLUTinhibitor cytochalasin B. Moreover, results showed that GLUE1 is unableto transport 3-O-methylglucose. Through the use of sensors expressedinside the lumen of the endoplasmic reticulum, it could be demonstratedthat GLUEs not only mediate uptake into the cytosol, but can also exportglucose out of the cytosol. Similar results were obtained for severalArabidopsis paralogs of this gene family.

In order to exclude the possibility that GLUE1 interacts with anendogenous signaling cascade, GLUE1 was expressed in aglucose-uptake-deficient yeast strain EBY4000. Results showed that GLUE1mediates uptake of glucose with a K_(M) of 10 mM in anenergy-independent manner. GLUE1 encodes a small protein that containsseven transmembrane spanning domains, similar to the water and solutetransporting aquaporins. GLUE1-GFP fusions localize to the plasmamembrane (see, e.g., FIG. 9).

Taken together, these data show that GLUE1 encodes a novel class ofsugar uniporters, with properties identical to the root transport systemin Arabidopsis roots (Chaudhuri et al. Plant J. 56:948-962, 2008).Analysis of microarray data shows that members of the family are indeedexpressed in roots.

Members of this protein family are expressed for example in nectaries(Ge et al. Plant J. 24:725-734, 2000) and in the tapetum (Guan et al.Plant Physiol. 147:852-863, 2008). Given their ability to efflux sugars,GLUEs may be responsible for the secretion of glucose to produce nectarin flowers, that they export glucose from the tapetum to supplydeveloping pollen and secrete glucose into the rhizosphere to attractand feed beneficial microorganisms (Chaudhuri et al. Plant J.56:948-962, 2008).

Example 5 A Human Homolog of the Plant GLUE Transporters

Similarity searches identified a GLUE homolog in the mouse and humangenome named RAG1AP1 (Tagoh et al. Biochem Biophys Res Commun221:744-749, 1996). RAG1AP1 shares significant homology with the plantGLUEs and also encodes a protein with seven predicted transmembranespanning domains. In contrast to Arabidopsis, the mouse and humangenomes each contain only a single member. A mutant lymphocyte cell linelacking RAG1AP1 activity was shown to control the expression of genesinvolved in antibody maturation (Tagoh et al. Biochem Biophys Res Commun221:744-749, 1996). This may be an indirect effect caused by theinability to secrete glucose or glucose analogs. RAG1AP1 may have anindirect role in controlling the expression of genes involved inantibody variation. Microarray studies demonstrate that this gene ishighly induced during lactation (FIG. 9).

Together with the functional evidence that the plant GLUEs are involvedin the secretion of glucose in plants, RAG1AP1 may function in a roleeither in lactose secretion and/or in glucose transport in the alveolarcells of the mammary gland. This is supported by data from a largeproteomics program, the Human Protein Atlas, which suggests that RAG1AP1(RAG1 activating protein 1) is specifically expressed in glandular cellsof the breast(www.proteinatlas.org/normal_unit.php?antibody_id=18095&mainannotation_id=1747078).Moreover, the protein appears to localize also to other glandular cellsin the human body, e.g. in the epididymis, potentially feeding spermcells.

Example 6 Plant Sugar Efflux Transporters for Nutrition of Pathogens

Materials and Methods

qPCR and RT-PCR Analysis.

Total RNA was extracted from HepG2 or HEK293T cells using an RNeasy MINIkit (QIAGEN, Hilden), first strand cDNA was produced (New EnglandBiolabs) and fragments of the predicted length were obtained by RT-PCRusing a set of GLUT and SGLT primers published previously. Samples wereseparated on a 2% agarose gel. For samples inoculated by Pst DC3000,total RNA was extracted from the leaves using Trizol reagent(Invitrogen). Real-time quantitative PCR (qPCR) was performed usingHotStart-IT SYBR Green qPCR Master Mix (USB) according to themanufacturer's instructions on a 7300 PCR system (Applied Biosystems).Actin (ACT8) expression was used to normalize expression values in eachsample; relative expression values were determined relative to the valueof the sample infiltrated with 1 mM MgCl₂ buffer at each time pointusing the comparative 2^(−ΔΔCt) method. For samples infected by G.cichoracearum, qPCR assays were performed using a LightCycler® 480(Roche). For quantification, relative transcript levels for each genewere normalized to ACT8 following the 2^(−ΔΔCt) method. Fold-change wascalculated relative to the untreated sample. Analysis was repeated twiceindependently. Induction is confirmed by microarray data(Genevestigator).

Constructs.

Cloning of the SGLT1 ORF in pOO2 has been described. SWEET1, SWEET8 andOsSWEET11 ORFs were amplified by RT-PCR using specific primers fromArabidopsis and rice, respectively. First strand cDNA from rice waskindly provided by Pamela Ronald, UC Davis. The ORFs were cloned intopDONR221 (Invitrogen) by Gateway BP clonase reactions, and mobilizedinto the yeast expression vector pDRf1-GW by Gateway LR recombinationreactions. SWEET1 was cloned into p112-A1NE-GW for yeastco-transformation with FLII¹²Pglu700μΔ6 in pDRf1-GW. p112-A1NE-GW wasgenerated by inserting a Gateway cassette into the SmaI restriction siteof p112-A1NE. For radiotracer experiments, ORFs with stop codons forSWEET1, SWEET8 and OsSWEET11 were cloned into the oocytes expressionvector pOO2-GW (D. Logué, unpublished results) by Gateway LRrecombination reactions.

FRET Analysis.

Cell culture, transfection, image acquisition and FRET analysis wereperformed as described previously.

A modified version of the yeast strain EBY4000 (hxt1 through −17Δ::loxPgal2Δ::loxP stl1Δ::loxP agt1Δ::loxP ydl247wΔ::loxP yjr160cΔ::loxP)carrying a cytosolic invertase (YSL2-1) was transformed with SWEETs andHXT5 and grown on SD (synthetic deficient) medium supplemented with 2%maltose and required auxotrophic markers. For complementation growthassays, cells were grown overnight in liquid minimum medium to OD₆₀₀˜0.6and then diluted to OD₆₀₀˜0.2 using water. Serial dilutions (1×, 5×,25×, and 125×) were plated on SD media containing either 2% maltose (ascontrol) or 2% glucose and the relevant auxotrophic markers. Growth wasdocumented by scanning (CanoScan, Canon) the plates after 2-5 days at30° C.

Yeast uptake. Yeast cells were grown in SD medium supplemented 2%maltose and auxotrophic markers. Cells were harvested at OD₆₀₀ 0.5-0.7by centrifugation, and washed twice in ice-cold distilled water. Cellpellets were weighed after the supernatant had been removed. Cells wereresuspended 5-10% (w/v) in 40 mM potassium phosphate buffer, pH 6.0.Cells were pre-incubated in potassium phosphate buffer for 5 min at 30°C. For each reaction, 330 μl pre-warmed buffer containing 20 mM glucose(0.55 μCi D-[U-¹⁴C] glucose; 590 KBq/gmol, Amersham Pharmacia BiotechInc.) was added to an equal volume of cells. 120 μl aliquot werewithdrawn and transferred to the ice-cold water. Cells were harvested byvacuum filtration onto a glassfiber filters (GF/C, Whatman), and washedtwice in 10 ml ice-cold water. Filters were transferred to scintillationvials containing 5 ml of Ultima Gold XR Scintillator liquid (PerkinElmer). Radioactivity taken up by the cells was measured by liquidscintillation spectrometry. To determine substrate specificity forSWEET1 compared to D-glucose, a ten-fold excess of competing sugarspecies was used. To determine the pH-dependence of SWEET1 activity, 40mM potassium phosphate uptake buffer at specified pH was used. Threeindependent transformants were used for uptake experiments.

Xenopus Oocytes Isolation and RNA Injection.

After linearization of the pOO2 plasmids with MluI, capped cRNAs weresynthesized in vitro by SP6 RNA polymerase using mMESSAGE mMACHINE kit(Ambion, Inc., Austin, Tex.). Xenopus laevis oocytes were kindlyprovided by M. Goodman (Stanford University). Microinjection was carriedout as described by Ballatori et al. 25 ng to 50 ng of each cRNA wasinjected into healthy-looking oocytes (RNAse-free water was used ascontrol). The injected oocytes were then maintained at 18° C. inmodified Barth's saline (MBS: (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO₃, 0.82MgSO₄, 0.33 Ca(NO₃)₂, 0.41 CaCl₂, and 20 HEPES-Tris, pH 7.5) with 100 μMgentamycin, 100 U/ml penicillin and 100 μM streptomycin solution for 2-3d. The incubation buffer was changed once per day.

Tracer Uptake in Xenopus Oocytes.

The assay was performed with modification as described in Detaille etal. Two days after injection, groups of 7 to 16 oocytes were transferredinto tubes containing 200 μL MBS and 1 mM D-glucose (4 μCi/mlD-[¹⁴C(U)]-glucose; 319 mCi/mmol, PerkinElmer). After incubation at 20°C. for one hour, and the cells were washed by adding 1 ml ice-cold MBS.Incubation was stopped by adding ice-cold MBS buffer. The ooctyes werewashed three times in ice-cold MBS buffer. The cells were solubilizedwith 100 μl 1% (w/v) SDS, and measured individually.

Tracer Efflux Assay in Xenopus Oocytes.

Efflux was measured essentially as described. Three days after cRNAinjection, oocytes were injected with 50 nl solution containing 10 mMD-glucose with 0.18 μCi/μl D-[¹⁴C(U)]-glucose. Cells were immediatelywashed once in MBS. At defined time points, the reaction buffer (950 μl)was removed for scintillation counting and replaced with fresh medium.Finally, the oocytes were solubilized with 1% SDS and analyzed forradioactivity.

Analysis of Glucose Accumulation in Yeast Cells by FRET Sensors.

FRET measurements in yeast cells were performed as described.

Plant Growth and Pathogen Infection.

Arabidopsis Col-0 plants were grown in growth chambers under 8 hlight/14 h dark at 22° C. Five-week-old leaves were infiltrated with a 1mM MgCl₂ buffer, 2×10⁸ cfu/ml Pst DC3000 or Pst DC3000 ΔhrcU suspensionsin 1 mM MgCl₂ using needleless syringes. Leaf samples were collectedafter 6, 12, and 24 h incubation in the light. G. cichoracearuminoculation was performed as described. Plants were placed in a“settling tower” (cardboard box) and Arabidopsis plants were inoculatedwith G. cichoracearum spores by holding infected squash leaves over thesettling tower and using compressed air (duster cans) to blow the sporesoff of the squash leaves for settling onto Arabidopsis plants. Theinoculum density was ˜25-35 conidiospores/mm⁻². After inoculation,plants were incubated for 1 h in a dark dew chamber, then transferred toa growth chamber at 16 h day length and 70% relative humidity.

Alignment and Phylogenetic Analysis.

Multiple alignment of SWEET amino acid sequences was performed withCLUSTALW using default parameters, and a phylogenetic analysis wasperformed using the software Mega V3.1. Bootstrapping was performed 1000times to obtain support values for each branch. For pair-wisecomparison, multiple alignments of complete amino acid sequences wereconducted using the Vector NTI advance 11.0.

Confocal Microscopy.

Imaging of plants expressing YFP::SWEET1 and YFP::SWEET8 was performedon a Leica TCS SP5 microscope. YFP was visualized by excitation with anargon laser at 514 nm and spectral detector set between 525 and 560 nmfor the emission. The specimen were observed with 40/0.75-1.25NA HCX PLAPO CS objective.

Results and Discussion

Sugar efflux is an essential process required for cellular exchange ofcarbon skeletons and energy in multicellular organisms and ininteractions between organisms. Sugar efflux from the tapetum ortransmitting tract of the style fuels pollen development and later onpollen tube growth. Flowers secrete sugars for nectar production toattract pollinators and plants secrete carbohydrates into therhizosphere, potentially to feed beneficial microorganisms. Sugar effluxcarriers are required at many other sites, including the mesophyll inleaves and the seed coat. The molecular nature of the effluxtransporters is unknown. Plant-derived sugars also provide a substratefor pathogens. The primary goal of pathogens is to access nutrients fromits host plant to efficiently reproduce. Phytopathogenic bacteria in thegenera Pseudomonas and Xanthomonas can live in the extracellular space(apoplasm) of plant tissue, where they acquire carbohydrates as theirsource of energy and carbon skeletons. Successful pathogens likelyco-opt such mechanisms to alter nutrient flux. As a consequence,pathogens and plants engage in an evolutionary tug-of-war in which theplant tries to limit pathogen access to nutrients and initiates defensestrategies, while the pathogen devises strategies to gain access tonutrients and suppress host immunity. Insight to the mechanisms used bypathogens to alter plant defenses is now emerging; however, little isknown about how pathogens alter host physiology, notably sugar export,to support pathogen growth. We thus postulated the existence oftransporters, either vesicular or at the plasma membrane, that secretesugars. We also hypothesized that these plant efflux transporters are‘co-opted’ by pathogens to supply their nutrient requirements. At leastin the case of wheat powdery mildew, glucose is the main sugartransferred from plant host to pathogen. Respective pathogen glucose/H⁺cotransporters have been identified; in contrast, the plant sugar effluxmechanisms have remained elusive.

To identify novel glucose transporters from the reference plantArabidopsis, genes encoding uncharacterized polytopic membrane proteinsfrom the plant membrane protein database Aramemnon, available on theinternet at “aramemnon.botanik.uni-koeln.de”, were screened using amammalian expression system. Candidate genes were coexpressed with ahigh-sensitivity FRET glucose sensor (i.e. FLIPglu600μΔ13V) in humanembryonic kidney HEK293T cells, which are characterized by lowendogenous glucose uptake activity. Among the genes tested, SWEET1(AT1G21460) expression enabled HEK293T cells to accumulate sugars asdetected by glucose-dependent negative FRET ratio change; consistentwith a transport function (FIG. 9A). To determine whether SWEET1 canalso mediate efflux from the cytosol, we expressed the FRET glucosesensor FLIPglu600μΔ13V^(ER) in the lumen of the endoplasmic reticulum(ER; FIG. 9B). Topologically, uptake across the plasma membrane (PM) isinitiated from the extracellular side of the carrier, while ‘export’ tothe ER is initiated from the cytoplasmic side of the transporter (FIG.9C). The glucose-dependent response of the ER sensor demonstrates thatSWEET1 can mediate uptake across the PM and ‘efflux’ into the ER. SWEET1may thus function as a glucose uniporter, for which the direction oftransport depends only on the glucose gradient across the membrane.Endogenous glucose transporters (GLUTs) in HEK293T cells were notinvolved in glucose uptake because the GLUT inhibitor cytochalasin B didnot affect SWEET1-induced glucose uptake (FIG. 12A). Furthermore, themRNAs levels of known human glucose transporters in the GLUT and SGLTfamilies were not induced in HEK293T cells expressing SWEET1 (FIG. 12B).To independently demonstrate that SWEET1 activity is required forglucose uptake, SWEET1 was expressed in a yeast mutant lacking all 18hexose transporters. SWEET1 enabled the yeast mutant to grow on glucose(FIG. 9D) and to accumulate intracellular glucose as determined usingthe FRET glucose sensor FLII¹²Pglu700Δ∂6 (FIG. 9E). Furthercharacterization revealed that SWEET1 functions as a low affinitytransporter in yeast with a K_(m) for glucose of 9 mM (FIG. 9F).Consistent with a uniport transport mechanism, uptake was not stimulatedby energization, and was largely pH-independent (FIG. 13A). Similar tothe glucose transport activity described in Arabidopsis roots,SWEET1-mediated uptake was marginally inhibited by the glucose analog3-O-methylglucose (FIG. 13B). In support of a role in cellular uptakeand efflux, a constitutively expressed SWEET1-YFP fusion localizes tothe PM in Arabidopsis leaves (FIG. 9G). Based on microarray studies,SWEET1 is only weakly expressed in roots, but highly expressed inArabidopsis flowers, suggesting a role in supplying nutrients to thegametophyte or nectaries (FIG. 14). Despite the striking similarity ofthe biochemical properties of a putative sugar transporter in the rootsystem, SWEET1 expression in roots is low, suggesting that it does notplay a major role glucose efflux from roots. Other proteins, possiblyrelated to SWEET1, may be involved in sugar transport in roots.

SWEET1 is the first characterized member of a novel transporter family(PFAM PF03083) with 17 members in Arabidopsis and 19 in rice (FIG. 1).Arabidopsis SWEETs are diverse, falling into four subclades (FIG. 1A)with identities ranging between 28 and 86% (Tables 1 and 2). Consistentwith functions in transport, SWEETs are small hydrophobic proteinspredicted to form a hydrophobic pore built by 7 transmembrane helices(TMH). In silico analysis suggests that the 7 TMHs in SWEETs resultedfrom an ancient duplication of a 3-TMH domain polypeptide (1-3 and 5-7)fused via TMH 4 (FIG. 9H).

While none of the members of this family had been characterizedfunctionally, phenotypes of several SWEET mutants have been described.SWEET1 is 41% identical to its paralog SWEET8, and belongs to the secondof the four Arabidopsis SWEET clades. Mutation of SWEET8/RPG1 had beenshown to lead to male sterility. Coexpression of SWEET8/RPG1 with theFRET sensors FLIPglu600μΔ13V or FLIPglu600μΔ13V^(ER) in HEK293T cellssuggests that SWEET8 also functions as a uniporter (FIG. 15A; FIG. 9C).Moreover SWEET8/RPG1 complements the yeast glucose transport mutant(FIG. 9D). SWEET8/RPG1 is expressed the tapetum, strongly suggesting arole as a glucose effluxer necessary for pollen nutrition.

SWEET1 and SWEET8 share 34% amino acid sequence identity with the riceprotein OsSWEET11/Os8N3 (named OsSWEET11 based on phylogeny, FIG. 1).The closest Arabidopsis homolog shares 40% identity with OsSWEET11/Os8N3and belongs to the third SWEET clade (FIG. 1). Similar to SWEET8,OsSWEET11/Os8N3 appears to function in pollen nutrition since areduction of its expression by RNA-inhibition led to reduced starchcontent in pollen as well as pollen sterility. Silencing of PetuniaNec1, another homolog of SWEETs in clade 3 (FIG. 1) also led to malesterility. Nec1 is expressed in nectaries, and its developmentalregulation correlated inversely with starch content of the nectaries,suggesting a second role for Nec1 in sugar secretion in nectaries. Takentogether, these data strongly suggest that in both mono- anddicotyledonous plants SWEETS play a crucial role in supplyingcarbohydrates to key reproductive purposes.

Pathogens use the host plant's photosynthetic capacity to provide energyand nutrients to grow and reproduce. It has been well established that awide variety of pathogens acquire glucose from their hosts. It washypothesized that different pathogens highjack the host sugar effluxsystems dedicated for plant development, such as feeding of thegametophyte. Accordingly, it was then tested whether the mRNA levels ofArabidopsis SWEET family members are altered by bacterial and fungalpathogens (FIG. 10). Pseudomonas syringae pv. tomato strain DC3000infection highly induced SWEET4, 5, 7, 8 and 15 mRNA levels inArabidopsis leaves. In contrast, the DC3000 type III secretion mutant(ΔhrcU), which cannot inject type III effector proteins into the hostand is thus compromised in pathogenicity, did not induce four of thefive genes demonstrating that SWEET mRNA abundance is modulated in atype III-dependent manner. It was then tested whether other pathogenstarget the same or different family members. The powdery mildew fungusGolovinomyces cichoracearum induced a different set of SWEET mRNAs, mostprominently SWEET12 (FIG. 10A, C). Microarray data showed that thefungal pathogen Botrytis cinerea targets again a different set of SWEETs(i.e. SWEET4, 15, 17). Taken together, pathogen-specific modulation ofSWEET mRNA levels likely alters sugar transport at the site of infectionimpacting pathogen growth and plant immunity.

Consistent with this hypothesis, the rice gene OsSWEET11/Os8N3, which isimportant for pollen nutrition, functions as a pathogen susceptibilityfactor. The rice ossweet11/os8n3 mutant was found to be resistant to thebacterial pathogen Xanthomonas oryzae pathovar oryzae (Xoo) strainPXO99^(A), strongly suggesting that OsSWEET11/Os8N3 supplies sugars tothe pathogen during infection (FIG. 11A). Accordingly, it was testedwhether OsSWEET11/Os8N3 also functions as a glucose transporter. UnlikeSWEET1 and SWEET8, OsSWEET11/Os8N3 did not mediate glucose uptake inHEK293T cells and did not complement the yeast hexose transport mutant(data not shown), indicating that it does not function in glucoseuptake. It was however conceivable that OsSWEET11/Os8N3 functions as aglucose effluxer. To test this hypothesis, OsSWEET11/Os8N3 was expressedin Xenopus oocytes, a system amenable for efflux studies. OsSWEET11, incontrast to SWEET1 and the mammalian Na⁺-dependent glucose transporterSGLT1, was also not able to mediate [¹⁴C]-glucose uptake into oocytes(FIG. 9I; FIG. 16). However, coexpression of SGLT1 and OsSWEET11/Os8N3led to reduced [¹⁴C]-glucose accumulation in the oocytes, a findingcompatible with an efflux (i.e. ‘leak’) activity of OsSWEET11/Os8N3(FIG. 9I, K). The hypothesis that OsSWEET11/Os8N3 can export glucose iscorroborated by direct efflux measurements. Glucose efflux was measuredby injecting [¹⁴C]-glucose into oocytes expressing the plant proteins.SWEET1 and OsSWEET11/Os8N3 were both able to efflux [¹⁴C]-glucose (FIG.9G), suggesting that while SWEET1 functions as a facilitator,OsSWEET11/Os8N3 is an effluxer (potentially a H⁺/glucose antiporter).Moreover, OsSWEET11 (Os8N3) can transport sucrose (FIG. 43G). Thus,OsSWEET11/Os8N3 appears to be recruited by the pathogen to provideglucose and sucrose for reproduction.

The finding that OsSWEET11/Os8N3 functions as a sugar effluxer providesa model of how pathogens co-opt basic plant function to gain access tothe plant's energy resources (FIG. 11). Xoo strain PXO99^(A) depends onthe type III effector gene pthXo1 for infection of rice. PthXo1 is a TAL(transcriptional activator-like) effector, which directly interacts withDNA to promote transcription of target genes. PthXo1 secreted by XooPXO99^(A) specifically activates transcription of OsSWEET11/Os8N3,presumably to induce sugar efflux in order to feed the apoplasmicbacteria (FIG. 11A). When PthXo1 is mutated (ME), transcription ofOsSWEET11/Os8N3 and pathogenicity are reduced, consistent withstarvation of the pathogen (FIG. 11B). If OsSWEET11/Os8N3 becomesunavailable due to mutation or RNA inhibition, sugar (e.g. glucose, glc)would not be exported in sufficient amounts and the pathogen wouldstarve (FIG. 11C). Indeed, ossweet11/os8n3 mutants are resistant to XooPXO99^(A). PXO99^(A) bacteria carrying another TAL effector (AvrXa7) arevirulent even in the ossweet11/os8n3 mutant (FIG. 11D), compatible withthe most parsimonious hypothesis that other SWEETs are co-opted by thepathogen to support bacterial growth (FIG. 11D). Indeed, the predictedDNA sequence targeted by PthXo1 is TRCA•CT•CCATTACTRTAAAA•N• (SEQ ID NO:125)(found in the promoter upstream of OsSWEET11/Os8N3), whereas thattargeted by AvrXa7 is TA•AANCRCCCN••CCNNRRATRA•N (SEQ ID NO: 126). Thissequence was not sufficient to identify the potential targets. Thesefindings support the notion that besides their role in immunity, typeIII effectors are also involved in providing access to nutritionalresources of the host plant. How fungal pathogens target promoters ofthese transporters is not understood yet, however the transporter genesmay be suitable diagnostic tools to unravel the regulatory networkssupporting fungal growth. Apparently, in order to be maintained inevolution despite this high pathogen-based selection pressure, SWEETtransporters must have essential functions in the plant; the analysis ofmutants suggests that at least one of them plays a role in supplyingcarbohydrates to the gametophyte. Thus, the activities of the otherparalogs may also be critical to other plant functions. Characterizationof the remaining SWEET paralogs and analysis of mutants especially withregard to disease susceptibility will be important next steps.

Knowledge of the full spectrum of pathogen effector molecules and howthey disrupt plant metabolism to favor pathogen growth will improve ourunderstanding of host-pathogen interactions and may lead to newstrategies for combating pathogen infections, which at the global scalelead to crop losses of over 10% annually. Moreover, analysis of theother genes in the SWEET family may help solve some of the riddles ofpollen nutrition, nectar production and carbon sequestration from plantroots. Interestingly, animal genomes contain SWEET homologs alsoinvolved in sugar transport.

Example 7 A Third Family of Glucose Transporters in C. elegans andHumans

Materials and Methods

The ORF of SGLT1 (Invitrogen, Carlsbad, Calif.) was amplified by PCR andcloned into pCR2.1-TOPO (Invitrogen). The SGLT1 ORF was excised withEcoRI/XhoI and cloned into the corresponding sites in pOO2. The splicevariant RAG1AP1-1 in pDNR-LIB (Clone ID: 4076256, Open Biosystems,Huntsville, Ala.) was restricted with EcoRI/XhoI and cloned into pOO2.RAG1AP1-2 in pCMV-SPORT6 (Clone ID: 3896154, Open Biosystems,Huntsville, Ala.) was transferred into the pOO2-GW (D. Logué,unpublished) by in vitro LR recombination (Gateway). RAG1AP1-3aa wasmutated to Y216A, L218A, L219A (putative internalization motifs) bysite-directed mutagenesis and cloned by in vitro BP recombination inpDONR221-f1 and further mobilized into pOO2-GW by LR reaction. CeSWEET1(K02D7.5), CeSWEET3 (C06G8.1), CeSWEET4 (Y39A1A.8), CeSWEET5 (K06A4.4),and CeSWEET7 (K11D12.5) (Open Biosystems, Huntsville, Ala.) were clonedinto pOO2-GW using LR reactions. Subsequently, a start and stop codonwas added using site directed mutagenesis. Oocyte expression andtransport studies were performed as described by Chen et al, except thatfor all uptake experiments oocytes were preincubated in 1 mM glucose inMBS overnight.

Results and Discussion

The C. elegans genome contains 7 homologs of a novel class of sugarefflux transporters (SLC50), while the human genome has a singlehomolog, named RAG1AP1. Similar to the Arabidopsis SWEET1, C. elegansCeSWEET1 mediates glucose uptake when expressed in Xenopus oocytes.CeSWEET1 as well as human RAG1AP1 counteract secondary active glucoseaccumulation in oocytes mediated by the Na⁺/glucose cotransporter SGLT1.Mutation of CeSWEET1 led to fat accumulation, compatible with a defectin cellular glucose efflux leading to accumulation of lipids. Thesefindings may shed new light on a role of the human homolog RAG1AP1 insugar transport.

The human genome contains at least two classes of glucose transporters,SLC2 and SLC5. SLC2, named GLUTs are uniporters, i.e. they transportglucose along its concentration gradient. In contrast, SGLTs areNa⁺-coupled cotransporters that can actively import glucose driven by asodium gradient. These transporters can explain most of the uptakeactivities found in humans, e.g. a GLUT2 mouse knock-out mutant showsdramatically reduced uptake capacity. However, surprisingly, glucoseclearance was normal, suggesting the existence of an alternative effluxroute. The use of a FRET sensor based expression cloning system lead tothe identification of a novel class of glucose transporters in plants(accompanying ms). Arabidopsis SWEET1 and 8 function as uniporters,while the rice OsSWEET11 appears to efflux glucose and sucrose.Bioinformatic analyses showed that animals and human genomes containhomologs, registered as solute carrier family SLC50(www.bioparadigms.org/slc/intro.htm) (FIG. 1). Here we show that the C.elegans CeSWEET1 mediates weak, but significant glucose uptake whenexpressed in Xenopus oocytes (FIG. 19B). The activity is significantlyweaker than that of SGLT1 or the plant homolog SWEET1. In comparison,the C. elegans homologs CeSWEET3, 4, 5 and 7 did not mediate detectableglucose uptake in oocytes (FIG. 19B). The plant homolog OsSWEET11, whicheffluxes glucose and sucrose, had been shown to counteractSGLT1-mediated glucose accumulation in oocytes when coexpressing bothproteins. The working hypothesis is that OsSWEET11-mediated efflux actsas a ‘leak’, preventing accumulation of glucose in the oocyte (3). Totest whether the C. elegans homologs may potentially function aseffluxers, we coexpressed them with SGLT1 (FIG. 19C). All five homologstested lead to reduced glucose accumulation as compared to SGLT1 alone.Direct efflux measurements from oocytes injected with [¹⁴C]-glucose showthat RAP1AP1 induces efflux of glucose from oocytes (% glucose releasedwithin 2 min: RAG1AP1: 5.27±0.29; control: 0.91±0.23; S.E.; n>7).Mutations in one of the C. elegans homologs (CeSWEET1; K02D7.5) leads tofat accumulation, consistent with a lack in the ability to effluxsugars. Similar to CeSWEET3, 4, 5 and 7, the human homolog RAG1AP1(renamed HsSWEET1) was also unable to mediate glucose accumulation inoocytes, but counteracted SGLT1-mediated glucose accumulation. This wastrue for the two splice variants and a mutated version carrying threemutations in three residues forming putative internalization motifs(FIG. 19C). Mutations in the homolog Ci-Rga (CiSWEET1) from the seasquirt Ciona lead to early developmental defects, underlining theimportance of these genes for metazoa. In mammals, at least one of theglucose efflux routes from liver has remained elusive.

RAG1AP1 had been named Recombination Activating Gene 1 ActivatingProtein 1 since a defect in a cell line affected recombination.Moreover, the gene has been named RGA and has been implied in targetingof TRPV ion channels. It will be interesting to test the hypothesis thatRAG1AP1 may contribute to glucose efflux in liver.

Example 8 SWEET Sucrose Exportation in HEK293 Cells

SWEETs 1-4 and 6-11 and 14-16 were expressed in HEK293 with a FRETsucrose sensor. Several members of the SWEET family exported sucrose.The SWEET members that demonstrated this ability all belong to the sameclade (see FIG. 1). Accordingly, this clade appears to demonstrate astrong ability to export sucrose. OsSWEET11 belongs to that clade, thusbesides exporting glucose, these proteins also export sucrose. This maybe significant in targeting pathogens like Ustilago that take upsucrose. It has been reported that a novel high-affinity sucrosetransporter is required for virulence of the plant pathogen Ustilagomaydis. PLoS Biol. 8(2):e1000303) or cell wall invertase then cleavessucrose and the pathogen imports glucose.

The demonstrated ability of SWEETs to export sucrose is important as ithas long been known that sucrose effluxers are required for cell to celltransport in leaves, but the identity of these proteins has remainedelusive. As SUTs take up sucrose from the cell wall, somewhere in theleaf, sucrose produced in mesophyll cells has to efflux into the cellwall. The ability of SWEETs to export sucrose accordingly providessignificant data in understanding how plants achieve this. Moreover, thematernal tissue of a plant must export sucrose to supply developingseeds with sucrose as the main transported sugar in the plant. Thus, therole of SWEETs as sucrose exporters may be significant in developingseeds.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

The invention claimed is:
 1. A method of inhibiting the expression or activity of a SWEET transporter in a plant cell, the method comprising transforming the plant cell with an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the plant cell coding for the SWEET 12 transporter, wherein said dsRNA reduces sugar efflux out of the plant cell, wherein the plant cell is from or in soybean, tomato, alfalfa, potato, pea, wheat or maize, wherein the reduction in efflux of sugar out of the plant cell increases pathogen resistance of the plant cell to Golovinomyces cichoracearum.
 2. The method of claim 1, wherein the SWEET transporter protein is located in the cell membrane of the plant cell.
 3. The method of claim 1, wherein said sugar is glucose.
 4. The method of claim 1, wherein said sugar is sucrose.
 5. A plant cell comprising an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the plant cell that codes for a SWEET 12 transporter, wherein said dsRNA reduces sugar efflux out of the plant cell, wherein the plant cell is from or in soybean, tomato, alfalfa, potato, pea, wheat or maize, wherein the reduction in efflux of sugar out of the plant cell increases pathogen resistance of the plant cell to Golovinomyces cichoracearum.
 6. The plant cell of claim 5, wherein the SWEET transporter protein is located in the cell membrane of the plant cell.
 7. The plant cell of claim 5, wherein the sugar is glucose.
 8. The plant cell of claim 5, wherein the sugar is sucrose.
 9. The plant cell of claim 5, wherein the plant cell is in a plant or part thereof.
 10. The plant cell of claim 9, wherein the nucleic acid that inhibits the expression of the SWEET transporter in the plant cell is expressed in a part of the plant selected from the group consisting of root, stem, leaf, seed, flower, fruit, anther, nectary, ovary, petal, tapetum, xylem and phloem.
 11. A method of inhibiting the expression or activity of a SWEET transporter in a rice plant cell, the method comprising transforming the rice plant cell with an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the rice plant cell coding for a SWEET 12 transporter, wherein said dsRNA reduces glucose efflux out of the rice plant cell, wherein the reduction in efflux of sugar out of the plant cell increases pathogen resistance of the plant cell to Golovinomyces cichoracearum.
 12. The method of claim 11, wherein the SWEET transporter protein is located in the cell membrane of the rice plant cell.
 13. The method of claim 11, wherein the sugar is glucose.
 14. The method of claim 11, wherein the sugar is sucrose.
 15. A rice plant cell comprising an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the rice plant c cell that codes for a SWEET12 transporter, wherein said dsRNA reduces glucose efflux out of the rice plant cell, wherein the reduction in efflux of sugar out of the plant cell increases pathogen resistance of the plant cell to Golovinomyces cichoracearum.
 16. The rice plant cell of claim 15, wherein the SWEET transporter protein is located in the cell membrane of the plant cell.
 17. The rice plant cell of claim 15, wherein the sugar is glucose.
 18. The rice plant cell of claim 15, wherein the sugar is sucrose.
 19. The rice plant cell of claim 15, wherein the expression vector is expressed in a part of the plant selected from the group consisting of root, stem, leaf, seed, flower, fruit, anther, nectary, ovary, petal, tapetum, xylem and phloem.
 20. A method of inhibiting the expression or activity of a SWEET transporter in a plant cell, the method comprising transforming the plant cell with an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the plant cell coding for the SWEET 12 transporter, wherein said dsRNA reduces sugar efflux out of the plant cell, and wherein the plant cell is from or in soybean, tomato, alfalfa, potato, pea, wheat or maize.
 21. A plant cell comprising an expression vector, the expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the plant cell that codes for a SWEET 12 transporter reduce sugar efflux out of the plant cell, and wherein the plant cell is from or in soybean, tomato, alfalfa, potato, pea, wheat, rice, or maize.
 22. A method of inhibiting the expression or activity of a SWEET transporter in a plant cell, the method comprising: transforming the plant cell with an expression vector comprising a nucleic acid sequence coding for one or more polynucleotides that form a double-stranded RNA (dsRNA) to inhibit a nucleic acid sequence in the plant cell coding for the SWEET 12 transporter, wherein said dsRNA reduces sugar efflux out of the plant cell, and selecting a plant having reduced efflux of sugar such that resistance to Golovinomyces cichoracearum is increased, wherein the plant cell is from or in soybean, tomato, alfalfa, potato, pea, wheat or maize, and wherein the reduction in efflux of sugar out of the plant cell increases pathogen resistance of the plant cell to Golovinomyces cichoracearum. 